cord-007427-iqwojhq2	2019	Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents. Viral RNAs were examined for Lassa immediately after extraction by the staff of the Virology Laboratory of Hemorrhagic Fevers Research Project of Gamal Abdel Nasser University of Conakry, Guinea and were then used to assess diagnostic sensitivity and specificity. In addition, LOD was assessed using a series of 10-fold dilutions of ARPs. For this purpose, eight LASV sequences of a maximal number of mismatches in the targeting region of L gene were selected (including a sequence of the strain Josiah, which was also used for the generation of the positive controls) and generated for the production of ARPs as described above (Table 2 ) .
cord-007644-7bsixsgd	2000	authors: Chirnside, E.D.; Francis, P.M.; De Vries, A.A.F.; Sinclaira, R.; Mumford, J.A. title: Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus A recombinant glutathione-S-transferase fusion protein expressing amino acids 55–98 of equine arteritis virus (EAV) G(L) (rG(L)55–98) was tested in an ELISA for its ability to detect serum antibodies to EAV. This paper describes an indirect ELISA using a recombinant glutathione-Stransferase fusion protein (Smith and Johnson, 1988) as an antigen to screen equine sera for the presence of antibodies to EAV, and its evaluation as a diagnostic test with large numbers of equine serum samples. By testing > 1500 equine sera in ELISA to G,55-98, we have demonstrated that amino acid residues 55-98 of the Bucyrus strain of EAV G,_ encompass a highly immunoreactive antigen which correlates closely with the host virus neutralizing response.
cord-007648-tm0hn0hz	2002	Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The objectives of this work were to produce immunoadsorbents using monoclonal antibodies which have been prepared previously (Mockett et al., 1984) and to purify the virus-coded proteins of the viral envelope in a single step in relatively large amounts. In addition the sequential humoral antibody response of chickens after IBV infection has been studied using the purified viral proteins and whole virus in ELISAs and compared to the results using the neutralization test. Thc chicken, about 10 days after an IBV infection, has antibodies to both the spike and membrane proteins in its serum but only very low concentrations of neutralizing antibodies.
cord-015936-4fwkf8fn	2005	key: cord-015936-4fwkf8fn authors: nan title: SUBJECT INDEX, volumes 123-130 date: 2005-11-04 journal: J Virol Methods DOI: 10.1016/s0166-0934(05)00346-0 sha: doc_id: 15936 cord_uid: 4fwkf8fn nan HIV; 2 LTR circles; New marker (125) 11 HIV antigen/antibody combined assay; HIV; Serology; HIV-1 p24 antigen assay (127) (127) (128) (128) 67 Yeast expression; Pichia pastoris; SARS-CoV; N protein (130) 83 enzyme analysis; Flaviviruses; RT-PCR
cord-251974-2zwrqjj9	2009	The European Standard suggests other methods, based on gel-filtration techniques, using products as Sephadex TM LH-20, Microspin TM columns S400HR or Minicon ® concentrators, but they lead to problems of contact times, separation capacity and cost (supplementary data, Fig. 1 ). These tests were also performed with solutions of CHX and HXM at various concentrations and after their filtration on the "in-house" Sephadex TM columns to evaluate their retention and the elimination of potential cytotoxicity. Three types of controls were performed in each experiment to validate the results: (i) non-retention of viruses after filtration on Sephadex TM columns, (ii) neutralization of the potential antiviral activity of the product tested, and (iii) elimination of its cytotoxicity. To assess the non-retention of viruses, e.g. HCoV 229E, viral suspensions were mixed with sterile distilled water for the contact time defined for the experiment, filtered on the "in-house" Sephadex TM columns and titrated as describe above (Section 2.3).
cord-251991-ghbpga1s	2011	Respiratory syncytial virus (RSV) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (NHBE) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus–host interactions. Polarized Calu-3 are susceptible to RSV infection and release infectious virus primarily from the apical surface, consistent with studies in NHBE cells. The mechanisms of cellular responses to RSV infection have been studied extensively in vitro in a variety of immortalized epithelial cell lines grown in monolayer cultures, including but not limited to Vero, Hep-2, A549, and ଝ The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of CDC. Consistent with previous studies in polarized MDCK (Roberts et al., 1995) and in differentiated NHBE, polarized Calu-3 released infectious virus primarily from the apical surface, and infection was persistent, detectable for at least 6 weeks post-infection.
cord-252268-o63ep08b	2014	As this equation relies on consistency between samples in the RNA quantity assayed, and the optimum reaction efficiency, as well as minimal fluctuation in the expression of housekeeping genes (endogenous controls) (Peters et al., 2007; Mane et al., 2008; Bruder et al., 2010) , these parameters were optimized using RNA extracted from cultured ferret lymph node cells stimulated with mitogens or with influenza virus. To test the ability of ferret leukocytes to produce mRNA cytokines and chemokines, lymph node cells from naïve ferrets were cultured with various mitogens known to activate T and B lymphocyte and macrophage/monocyte responses (Fig. 5) and with live or heat inactivated influenza virus (Fig. 6 ) and the cytokine and chemokine expression profiles were determined (summarized in (ConA) or Phytohaemagglutinin (PHA), which act by cross linking T cell receptors via sugars on the surface of human T lymphocytes (Chilson and Kelly-Chilson, 1989) , induced similar cytokine profiles, increasing expression of IL2, IL4, IL6, IL10, IL17, Granzyme A, TNF␣ and IFN␥, most with high fold changes, consistent with effective stimulation of T lymphocytes (Fig. 5) .
cord-254210-3mi2aop5	2011	title: Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. In the present study, siRNAs were used for inhibition of the expression of the HTLV-1 structural genes gag and env. Forty-eight hours after transfection, the expression of EGFP-target (gag or env) fusion proteins was observed directly under an inverted fluorescence microscope. HEK 293 cells were observed 48 h posttransfection under a fluorescence microscope to determine the silencing effect of siRNAs on Gag and Env protein expression in cultured cells (Fig. 2B and C) . Based on the fluorescence data, flow cytometry and real time quantitative PCR, two siRNAs targeted to the HTLV-1 gag gene reduced efficiently gene expression by different amounts compared to the negative siRNA, scrambled siRNAs and controls without siRNA.
cord-255545-nycdhdsd	1999	In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. In fact, several studies have demonstrated that RT-PCR assays based on specific RNA template recognition by RT primers of defined polarity will not reliably distinguish between viral RNAs of positive or negative sense. Despite the findings above, many published research reports are based on conventional RT-PCR assays, relying on the polarity of primers added to the RT step in putative sense-specific measurements of viral RNAs; rarely are control reactions performed to rigorously show that this method is in fact sense-specific.
cord-255983-3dq99xz9	2012	The quantitative assay was compared to a commercial conventional multiplex PCR method (Seeplex TM RV detection kit, Seegene, Inc., Seoul, Korea) (Kim et al., 2009; Roh et al., 2008) respiratory samples from a study (Do et al., 2011) on acute respiratory infection in children at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. All samples were analyzed in parallel by the commercial multiplex Seeplex TM RV detection kit (Seegene, Inc., Seoul, Korea) according to the manufacturer''s instructions, to determine the presence of 12 respiratory viruses: human RSV subgroups A and B (RSV A, RSV B); influenza virus A (InfV A); influenza virus B (InfV B); human coronaviruses (229E, OC43), human metapneumovirus (hMPV), parainfluenza virus 1, 2 and 3 (PIV1, 2, 3), human rhinovirus (hRV A) and adenovirus (AdV) (Kim et al., 2009; Roh et al., 2008) and by the newly developed RSV LNA real-time RT-PCR.
cord-256355-muskjaw3	2002	A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan™ probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan™ probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. By combining RT-PCR with TaqMan™ genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential PCR-product carry over contamination. A hemi-nested reverse transcriptase PCR (hnRT-PCR) assay that uses a cocktail of primers capable of detecting the six established genotypes of rabies and rabies-related viruses has been described (Heaton et al., 1997) . Using the sequence data from a number of isolates from each of the rabies and rabies-related virus genotypes, TaqMan™ probes were designed to distinguish the six established genotypes of rabies and rabies-related viruses.
cord-256608-ajzk86rq	2019	An alignment search was performed with the default expectancy threshold value on all fasta files using primers and probes of the PCR test as search queries and the program SSEARCH available in the FASTA sequence analysis package (Brenner et al., 1998; Pearson, 1991; Pearson et al., 2017; . The in silico specificity is expressed as the percentage of specific hits of taxonomy classified sequences with a maximum of one mismatch per primer or probe as these are considered to be detected with the respective PCR test. To demonstrate the suitability of our in-house developed software tool PCRv, we determined the in silico sensitivity and specificity of three PCR tests for West Nile virus (WNV) recommended by the World Organisation for Animal Health (OIE) (Eiden et al., 2010; Johnson et al., 2001) .
cord-256845-5pjam7em	2017	In the case the sensitivity of the test might be ameliorated through further studies, the RT-LAMP could be extremely useful, due to its low costs and rapidity, in those situations where the Table 2 Results obtained on the samples tested with RT-nPCR (PCR) and LAMP and evaluated with agarose gel electrophoresis (LAMP GEL) and hydroxynaphtol blue dye (LAMP HNB detection of FCoV must be repeated over time and on a high number of cats (e.g. breeding catteries). Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis
cord-257284-dash9udv	2010	
cord-257785-jzdlvo7p	2010	The endpoint detection limit of the universal influenza A and H1N1 (2009) components of the triplex was directly compared to the published duplex using a dilution series of an influenza A H1N1 (2009) clinical sample and clinical samples containing seasonal H1N1 and H3N2 viruses. The endpoint detection limit of the H275Y component of the triplex was compared directly to the H275Y single assay using a dilution series of influenza A H1N1 (2009) H275Y positive clinical sample. Finally the ability of the triplex assay to detect minor populations of the oseltamivir resistant influenza A virus in samples containing a mixture of resistant and sensitive viruses was assessed. The endpoint detection limit of the universal influenza A and the H1N1/2009 component of the triplex assay in comparison to the duplex assay was assessed using serial dilutions of clinical samples containing influenza A H1N1 (2009) and seasonal H1N1 and H3N2 viruses (Table 3) .
cord-257850-x7qtxaum	2015	The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry. In two independent experiments, all ten influenza strains were correctly identified as either H1N1 or H3N2 influenza A virus, with an identification limit of 7 × 10 6 genome copies in the total sample volume subjected to LC-MS/MS analysis (corresponding to a CT value of approximately 30-32; Table 3 ). At this viral titer, the influenza viruses were typed and subtyped based on the identification of peptides derived from the nucleoprotein protein, indicated in boldface in Table 4 . The identification limit at which the RSV strain was identified correctly, was 3.6 × 10 7 genome copy equivalents in the total sample volume subjected to LC-MS/MS analysis (corresponding to a CT value of approximately 28; Tables 5 and S1).
cord-258008-t78svobg	2010	Two molecular assays were compared with real-time RT-PCR and viral culture for simultaneous detection of common viruses from respiratory samples: a multiplex ligation-dependant probe amplification (MLPA) and a dual priming oligonucleotide system (DPO). A panel of 168 culture-positive and negative samples was tested by the molecular assays for the presence of influenza A and B virus, respiratory syncytial virus, human metapneumovirus, rhinovirus, coronaviruses, parainfluenza viruses and adenovirus. Both molecular assays are comparable with real-time RT-PCR, more sensitive than viral culture and can detect dual infections easily. In this study, two commercial molecular assays, both designed for simultaneous detection of the most common viruses from a variety of respiratory samples, were compared with real-time RT-PCR and viral culture: a multiplex ligation-dependant probe amplification (MLPA) and a dual priming oligonucleotide system (DPO). Defined as true positives were samples that yielded positive viral detections by more than one method (culture, DPO, MLPA or real-time RT-PCR).
cord-258057-ti0rpt0q	2020	A loop-mediated isothermal amplification (LAMP) assay was established to detect PPV infection. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °C.The amplified products were analyzed both by visual inspection after staining with SYBR Green I dye and by conventional agarose gel electrophoresis. To verify the specificity of PPV detection by LAMP, the DNA (PPV, PCV2, PRV) and cDNA samples (PRRSV, CSFV) were amplified as sample templates by the LAMP reaction at 59.0℃ for 50 min and terminated at 80℃ for 3 min, respectively. The LAMP detection limit for PPV based on visual observation by addition of J o u r n a l P r e -p r o o f SYBR Green I and by gel electrophoresis analysis was 10 1 copies. Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification Rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (LAMP) method
cord-258468-52gej3co	2009	title: Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. In order to eliminate the cluster of rare codons from the 5 end of the cap gene, the expression vector encoding a truncated variant of Cap protein ( Cap-His), which was lacking the first 16 amino acid residues, was constructed (Fig. 1B) . In summary, a bacterial expression system has been developed for the production of the full-length recombinant capsid protein of porcine circovirus type 2.
cord-259212-pj8p2x9l	2003	
cord-259593-shrd1s7r	2007	title: siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA To study M protein function, three candidate small interfering RNAs (siRNAs) corresponding to M gene sequences were designed, transcribed in vitro, and then tested for their ability to silence M protein expression. The results showed that the mean green fluorescence intensity and M RNA transcripts were significantly reduced, and that the expression of M glycoprotein was strongly inhibited in those cells co-transfected with M-specific siRNAs. These findings demonstrated that the three M-specific siRNAs were able to specifically and effectively inhibit M glycoprotein expression in cultured cells by blocking the accumulation of mRNA, which provides an approach for studies on the functions of M protein and for the development of novel prophylactic or therapeutic agents for SCoV infection.
cord-259738-yuqc6dk0	2008	title: Enhancement of the immunogenicity of an infectious bronchitis virus DNA vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2 A DNA vaccine against infectious bronchitis virus (IBV) can induce specific humoral and cell-mediated immunity. When chickens were challenged with a virulent strain of IBV, the protective efficacy could be enhanced significantly after immunization with bicistronic DNA vaccine. In the present study, a bicistronic plasmid encoding the nucleocapsid protein and immune-stimulatory interleukin-2 has been constructed and its immunogenicity and protective effect in chickens has been evaluated. These recombinant plasmids were inoculated in chickens and tested in a protection-challenge experiment, demonstrating that vaccination with the co-expression plasmid pIRES-N/IL2 can induce stronger immune response than vaccination with pIRES-N. These results suggested that vaccination with the co-expression plasmid of N protein gene and IL-2 may have increased the protection rate against challenge. Partial protection against infectious bursal disease virus through DNA-mediated vaccination with the VP2 capsid protein and chicken IL-2 genes
cord-260208-fvdq0yes	2018	title: Rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. Six primers were needed in the RT-LAMP assays for TGEV, the reaction time was 30 or 60 min, and the results were visualized by agarose gel electrophoresis (Chen et al., 2010; Li and Ren, 2011 T Recombinase polymerase amplification (RPA) is an isothermal gene amplification technique that has been demonstrated to be a rapid, specific, sensitive, and cost-effective molecular diagnostic method (Daher et al., 2016; Piepenburg et al., 2006) . In this study, we developed and evaluated the userfriendly on-site detection platform integrating the real-time RPA technology and a field-deployable device (Genie III tube scanner) for the rapid detection of TGEV.
cord-260250-t48y27wg	2004	A TaqMan(®) fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 10(8) copies, allowing quantitation of samples with a wide range of CCoV RNA loads. As shown in Table 2 , the detec-tion limit of the TaqMan RT-PCR was 1-2 log higher than that of conventional RT-PCR, ranging around 10 1 copies/l and 10 −1.50 TCID 50 /50 l for standard RNA and CCoV strain, respectively, with a detection rate of 100% for each positive dilution. The results of the conventional amplification and real-time analysis carried out on the faecal samples of the CCoV experimentally infected dog are summarized in Fig. 3 .
cord-261089-aul4ifso	2016	The aim of this study is to develop a rapid, sensitive and specific duplex real-time RT-PCR assay for the simultaneous detection of TMEV and RTV, and provide a useful tool for the routine health monitoring of these two viruses in laboratory rodents and for the screening of contaminated biological materials. In addition, twenty cecum content and spleen samples collected from eight specific pathogen free (SPF) mouse strains (BALB/c, C57BL/6, DBA, FVB, 129, ICR, KM and NIH) and two rat strains (SD and Wistar) were used to evaluate specificity of the duplex real-time RT-PCR assay, these animals were reared under barrier colonies and were confirmed as serology negative for TMEV and RTV by a commercial ELISA kit (XpressBio, Maryland, USA). The specificity of the duplex real-time RT-PCR assay was determined by evaluation of RNA extracted from positive cultures of the following rodent viral pathogens: TMEV, RTV, MHV, Reo-3, RCV, Sendai, PVM, MNV and LCMV, and from twenty cecum content and spleen samples of ten SPF rodent strains.
cord-261134-zarq507s	2004	PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. These multiplex assays employ three primers; two consensus primers generate an amplicon diagnostic of an Old World Orthopoxvirus and the third primer simultaneously binds to a variola-specific polymorphism and initiates extension of a shorter PCR product to detect the presence of variola virus.
cord-261329-k1p7fo0e	2010	A rapid diagnostic method based on the melting curve SYBR Green I real-time PCR analysis was developed to detect and differentiate Newcastle disease virus (NDV) strains. The results obtained in this study demonstrate the possible applications for melting curve real-time PCR analysis in laboratory practice for the diagnosis and differentiation of avirulent and virulent strains of Newcastle disease virus. (2005) described a SYBR Green I real-time PCR melting curve analysis assay for differentiation, although the differences in the Tm values between the three genotypes were not very significant and could cause false characterization of the virus. Using the SYBR Green I real-time PCR melting peak analysis, it was possible to detect and differentiate virulent and avirulent strains of Newcastle disease virus. In this study, a method for the rapid detection and differentiation of Newcastle disease virus by SYBR Green I melting-curve analysis was described.
cord-261735-03hvi4el	2011	A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Frequently detected in tick-borne virus surveys in Africa (Guilherme et al., 1996) , DUGV is a tri-segmented single-stranded negative RNA enveloped virus and is considered endemic in arid regions (Burt et al., 1996) . The aim of this study was to develop a sensitive, specific and rapid one-step quantitative real time RT-PCR assay (qRT-PCR) to detect DUGV in infected cell supernatants, ticks or serum samples. The specificity was evaluated by using RNA extracted from supernatants from CCHFV, Hazara virus, Coronavirus and Influenza A virus infected cells. Among the 498 captured ticks, one Dugbe virus RNA was detected in one tick using the qRT-PCR assay (0.2%). In conclusion, a sensitive and specific qRT-PCR assay was developed to detect and quantify DUGV RNAs in infected cell supernatants, extracts from ticks and potentially sera.
cord-262991-j36vajdi	2002	
cord-263570-6notzm6s	2007	The load and distribution of CPV-2 mRNA in samples from infected dogs were estimated in comparison with the load of virus DNA, as evaluated by real-time PCR. The analytic specificity of spliced CPV-2 mRNA detection by real-time RT-PCR was assessed by testing RNA and DNA preparations of other canine pathogens, including canine coronavirus types I and II (CCoVI, CCoVII) (Decaro et al., 2005d) , canine distemper virus (CDV) (Elia et al., 2006) , canine adenovirus (CAdV) (Decaro et al., 2006a) , reoviruses (Decaro et al., 2005b) and rotaviruses . To compare the amount of viral DNA in the tissue samples and in the infected cell cultures, a real-time PCR assay was used as described previously (Decaro et al., 2005c) . The newly developed real-time RT-PCR assay was applied to determine the viral mRNA loads in different tissues of CPV-2 naturally infected dogs. A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 DNA in the feces of dogs
cord-264335-c2hfh3dq	2009	In order to detect and then type influenza A viruses most laboratories use a two tier testing system comprising of a universal influenza A screening assay complemented with a suite of subtyping assays that determine whether the sample is seasonal influenza A (human H1N1 and H3N2), avian H5N1 or the influenza A/H1N1/2009 virus. This article describes the development of a multiplex real-time reverse transcription polymerase chain reaction (rtPCR) that allows universal detection of all influenza A viruses and simultaneously subtypes all that are influenza A/H1N1/2009. Use of this assay will allow laboratories to screen respiratory samples for influenza A/H1N1/2009 virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. This article describes the development of a rapid, specific and sensitive multiplex rtPCR assay that detects all influenza A types and simultaneously identifies samples that contain the pandemic influenza A/H1N1/2009 virus.
cord-265634-7n4cvgs4	2002	title: Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry The current diagnostic methods for TSV and YHV include bioassay using indicator hosts, monitoring clinical signs, histopathology, dot blot, in situ hybridization using virus specific gene probe, immunohistochemistry and by the polymerase chain reaction (PCR) (Lightner and Redman, 1998) . SYBR Green RT-PCR was performed in a 96 well plate using 1 ml of each of the cDNA dilutions for TSV and YHV detection along with EF-1a and b-actin controls following the reaction parameters as described above. The analytical sensitivity of SYBR Green PCR was determined by using a serial dilution of TSV and YHV plasmid DNA as template for amplification. The SYBR Green RT-PCR was not only highly sensitive but also very specific for detecting TSV, YHV and the internal control genes, EF-1a and b-actin.
cord-266571-qbskh1uu	2002	Lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (PEDV) strain CV-777 and challenged 21 days later with the virulent isolate of the same virus. Virus-specific lymphoproliferative responses of systemic tissues (spleen and blood) and mesenteric lymph nodes were studied in conventional piglets after primary inoculation with the virulent, wild type, strain CV-777 of PEDV or its cell culture attenuated form and after challenge, 3 weeks later, with a high dose of the virulent virus. Correlations between lymphocyte proliferative responses in mononuclear cells collected from mesenteric lymph nodes, blood and spleen from pigs inoculated with virulent or attenuated PEDV or mock-inoculated and protection against challenge 21 days later with virulent PEDV.
cord-267588-ruuzr6l1	2020	This study aimed to examine the efficacy of six different swabs that are commonly found in hospital settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6" cotton tipped applicators, and HOLOGIC Aptima), along with more readily available alternative transport mediums (DMEM, PBS, 100% ethanol, 0.9% normal saline and VTM) for their use in molecular detection of SARS-CoV-2. Therefore, our results suggest that the cotton and wood For the portion of the study focusing on alternative transport media, we assessed the ability of DMEM, PBS, 0.9% Normal Saline, and 100% ethanol compared to VTM to be used as medium for the preservation and recovery of viral RNA to be quantified by molecular detection. Despite finding similar levels of viral RNA collected using different swabs and transport media, there is variation when evaluating different respiratory clinical samples while testing for SARS-CoV-2.
cord-267744-asjvf123	2007	Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5 × 10(7) and 9 × 10(6) transformants, respectively. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750–1000. The current study aimed to show that monoclonal IgY scFv antibodies which bind specifically to the S protein and SARS-CoV-infected Vero cells can be isolated from chickens immunized with Escherichia coli-derived S proteins. Cellular lysates containing single-chain variable fragment (scFv) antibodies from various Ssc (A) and Lsc (B) library clones were examined for their binding to SARS-CoV-infected cell lysates using a commercially available kit.
cord-267941-nrluar4e	2018	
cord-270421-ytrkob0h	2016	The CVB3 pseudovirus system was used for quantifying neutralizing antibody (NtAb) levels of 720 human serum samples and showed superior specificity and sensitivity comparing traditional cytopathic effect (CPE) assay. For the lack of CVB3 national antibody standard, we used a mouse serum collected from mice immunized with formalin inactivated CVB3 virus 112 strain and quantified its neutralizing activity against CVB3 (Nancy)-luc pseudovirus in duplicate on the same plate in six independent tests. We next used this pseudovirus assay to measure CVB3 neutralizing antibodies titers in serum samples collected from health adults (18-65 years old). In summary, we established a single round infection system of CVB3 and developed an in vitro assay for detecting neutralizing antibodies in clinical serum samples, and it was a superior surrogate of the assays using wild type viruses including traditional CPE assay and enzyme-linked immunosorbent spot assay.
cord-270526-o4hsr4pm	2007	The sensitivity and specificity of this assay were compared with a nested PCR assay by testing conjunctival swabs, nasal irrigation fluid, and blood lymphocyte samples from dogs suspected to have CD. The antemortem diagnosis of canine distemper is based on the demonstration of viral antigens in scrapings and body fluids such as conjunctival and vaginal smears, tracheal washings and Table 2 Clinical signs, age and breed of CD-positive specimens Table 3 Analysis of the sensitivity and specificity of the IC assay relative to nested PCR results in detecting CDV in specimens from dogs suspected to have CD a IC assay result One study using nested PCR analysis revealed that of 22 blood, 20 urine, 25 saliva, and 27 nasal swab samples from dogs suspected to have CD, 81.8%, 75%, 56%, and 70.3% tested positive, respectively (Shin et al., 2004) .
cord-270788-w0pewq52	2004	
cord-271915-nvilxnzl	2004	The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada. This single-tube RT-PCR is based on consensus primers targeting conserved regions of coronavirus genome sequences and allows for the detection and species identification of several coronaviruses including SARS-HCoV, with high analytical sensitivity. Aliquots of a 10-fold serial RNA dilution prepared from a lung biopsy sample of a patient with SARS (see Section 2) were used to compare our assay with the RealArt HPA coronavirus RT-PCR (Artus GmbH).
cord-273074-k8m917i4	2005	title: Preparation and evaluation of anti-SARS coronavirus IgY from yolks of immunized SPF chickens SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and neutralization test results showed that the IgY obtained was of a high purity and had a strong reactive activity with a neutralization titer of 1:640. In this study, we have successfully immunized specific pathogen-free (SPF) chickens, and then purified a high-titer anti-SARS coronavirus yolk immunoglobulin (IgY) with neutralizing activity against SARS coronavirus. The activity of IgY in sera and yolks diluted at 1:200 in phosphate buffer saline (PBS) from immunized animals was assessed using an indirect ELISA assay as described previously (Huang et al., 2005) (Fig. 1) . The development of high-titer anti-SARS coronavirus IgY described in this study would appear to have potential as a new anti-SARS biological product for passive immunization, as it effectively neutralized the SARS coronavirus.
cord-273846-l0elcfe8	2005	title: Effects of cold storage on detection of avian infectious bronchitis virus in chicken carcasses and local antibodies in tracheal washes In order to test the survivability of infectious bronchitis virus (IBV) in dead chicken carcasses during 24 h of cold storage, 7 week-old specific-pathogen-free chickens were infected with virulent IBV Massachusetts strain M41, and were killed humanely 10 days later. Trachea, lung, kidney and rectum were collected for virus isolation by tracheal organ culture (TOC) or embryonated chicken eggs (ECE), and detection by nested reverse-transcriptase polymerase chain reaction (RT-PCR). Diagnosis of infectious bronchitis virus (IBV) is confirmed by isolation of the virus using either chicken embryonated eggs (ECE) or tracheal organ culture (TOC) and detection by reverse-transcriptase polymerase chain reaction (RT-PCR) (Cavanagh and Naqi, 2003; Gelb and Jackwood, 1998) . The use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus
cord-274289-8g9tuyrc	2014	The study assessed the stability of nucleic acids stored on FTA cards at a temperature range representing the extremes of environmental heat (13 • to 46 • C) and cold specimen handling conditions (−7 • to −27 • C), and a timeframe from specimen collection to laboratory processing consistent with the expected extremes of diagnostic sample shipping (7-14 days). Bovine Coronavirus was the most prevalent virus detected by realtime PCR during this phase of the study, with 60% animals testing Table 2 Kappa values (95% CI) and percentage agreement for specimens collected into viral transport media compared to specimens collected onto FTA Cards. Bovine Respiratory Syncytial virus was detected using both the specimens in viral transport media and those on FTA Cards for 100% agreement; however the realtime PCR positive test result was for only one animal (Tables 1 and 2) .
cord-274954-06c3ymc3	2009	title: Development of a reverse transcription multiplex real-time PCR for the detection and genotyping of classical swine fever virus A reverse transcription multiplex real-time PCR (RT-MRT-PCR) was developed for rapid detection and genotyping of classical swine fever virus (CSFV). For the analytical sensitivity experiment, 100 samples of 14 CSFV genotype 1 strains and 86 samples from CSFV outbreak farms were all detected as CSFV-positive by RT-MRT-PCR, and the genotype results were consistent with the results of sequencing from a previous study. 10-fold serially diluted samples of C-strain, Q90-278, and 83-19 strains were determined to be CSFV-positive by viral isolation, RT-PCR, RT-nPCR, and RT-MRT-PCR. a A total of 169 clinical samples was detected for the presence of CSFV and the results included and used to evaluate agreement among viral isolation, RT-PCR, RT-nPCR, and RT-MRT-PCR. It was shown that the sensitivity of RT-MRT-PCR is comparable to those of RT-nPCR and viral isolation, and higher than RT-PCR for the samples of CSFV diluted serially (Table 4 ).
cord-275225-fvq8hezk	2012	The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale. The detailed analysis of the feline infectious peritonitis positive cat samples by SYBR-Green QPCR method revealed that the following organs harbour FCoV most frequently: lungs 6/6 (100%), liver 6/6 (100%), kidney 10/11 (90.9%), mesenteric lymph node 18/20 (90%), spleen 16/20 (80%) and gut 15/10 (66.7%).
cord-275787-5s442sy2	2016	title: Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). The parental cell line and clones were capable of expressing IFN beta and supported the replication of viruses such as vesicular stomatitis virus (VSV; family Rhabdoviridae, genus Vesiculovirus), herpes simplex virus (HSV; family Herpesviridae, subfamily Alphaherpesvirinae, genus Herpesvirus), PED-CoV and MERS-CoV. Bat kidney cells were immortalized by using ViaFect (Promega, USA) to transfect cells with either 2.5 g of pcDNA3 (Invitrogen, USA) empty vector or plasmids expressing either SV40 large T-antigen (SV40Tag) or Myotis polyomavirus large T-antigen (MyPVTag).
cord-275793-k0uvqcmp	2010	title: A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The objectives of this study were to develop a blocking microsphere-based immunoassay (bMIA) for detection of antibodies against BVDV, and to compare the performance of the assay with a commercial ELISA kit. This study describes the development and evaluation of a microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies. The diagnostic performance of the new bMIA was compared to that of a commercial blocking ELISA system, by testing a large panel of 509 bovine sera. A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk
cord-276368-c9e93h0u	2010	Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Table 1 List of primers used for conventional RT-PCR, nested PCR and SYBR Green real-time RT-PCR assay for the detection and quantitation of bovine and porcine toroviruses in the fecal specimens from diarrheic calves and piglets. The present study developed, optimized and validated a SYBR Green real-time RT-PCR assay using a universal primer pair for the detection and quantitation of both BToVs and PToVs in archived stool samples. The efficacy and reliability of the SYBR Green real-time RT-PCR assay for the detection and quantitation of BToV and PToV in the 121 bovine and 86 porcine stool samples were evaluated. In conclusion, a rapid, sensitive, specific and reproducible onestep SYBR Green real-time RT-PCR was developed for the detection and quantitation of BToV and PToV in bovine and porcine stool samples.
cord-276503-bh7uugwy	2009	Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Previous studies have focused on using flow cytometry to quantitate poliovirus infection in neuronal cells (Daley et al., 2005) , characterize enterovirus binding to host cell surfaces (Freistadt and Eberle, 2006; Mbida et al., 1991; Triantafilou et al., 2001) , confirm cytomegalovirus infection of tissue culture cells with a genetically engineered fluorescence reporter system (Kung et al., 2000) , and serotype human immunodeficiency virus type 1 (Zolla-Pazner et al., 1995) . In this study, flow cytometry proved to be a sensitive method for detecting fluorescently stained enterovirus-infected cells. False positives did not occur by flow cytometric analysis in that infected cells were negative after staining with isotype control antibodies and antibodies directed against other enterovirus serotypes.
cord-276541-u9ebql5a	2011	title: Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line Here, we constructed a single short hairpin RNA (shRNA) plasmid expression system that targeted the N gene and investigated whether shRNAmediated RNA interference could inhibit PHEV replication in PK-15 cells. To study the inhibitory effects of RNA interference on PHEV replication, the level of viral antigen produced in the PK-15 cells after shRNA transfection and viral infection was examined 48 h post-infection by an indirect immunofluorescence assay (IFA) using anti-PHEV serum. To quantify the effect of shRNA on viral replication at 48 h postviral infection, the viral genome copy number was determined by real-time PCR, using the serially diluted plasmid pT-N as a standard. It is clear from this study that the DNA vector-based shRNA approach, that is, the use of RNAi expression plasmids directed against the PHEV N gene, could effectively block expression of the viral target gene and inhibit viral replication.
cord-276718-3lujp0oy	2014	title: Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. In the present study, the RT-LAMP assay was developed for the detection and serotyping of DENV infection targeting the serotype specific regions of the NS1 gene using a real-time flourometer (Genie ® II from Optigene, U.K.). The performance of the RT-LAMP assay was validated by testing the samples simultaneously by the CDC real time PCR that is most sensitive and specific method for detection and differentiation of the DENV (CDC Dengue). Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay
cord-276739-84vf5bts	2011	Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. This method offers high sensitivity and selectivity, but generally requires approximately 2 h per run; therefore, qRT-PCR is not appropriate for rapid virus detection or subtyping in outbreaks of fast-spreading and/or highly pathogenic viruses at public health centers, hospitals, airports, and other public transportation hubs. The commercial reaction mixture was from the qRT-PCR kit, RNA-Direct TM SYBR ® Green Real-time PCR Master Mix (Toyobo, Osaka, Japan; http://www.toyobo.co.jp/e/). The SHRT-PCR system detects the highly conserved sequence of the corresponding viral genome, and the newly designed primer sets targeted for typing MP segments do not exhibit any cross reactions among other influenza viruses (Table 5 ).
cord-276989-441aclcc	2020	title: Development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific SIgA in colostrum On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Therefore, the aim of this study was to develop a simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled anti-SIgA SC mAb probe for the detection of anti-PEDV-specific SIgA in swine, and to compare its performance with an indirect SIgA ELISA based on the whole PEDV virus (Cong et al., 2019) . To test for colostrum, we diluted the samples 1: 20 in sample buffer and mixed thoroughly then, 100 μL of the solution was dispensed onto the sample pad well of the strip apparatus to determine the presence of PEDV specific SIgA, as described above.
cord-277057-ww41t4k2	2012	title: Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses() Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. This study reports the results of a comparison of the FTDRP multiplex assay with a panel of validated in-house singleplex real-time RT-PCR assays developed at the Centers for Disease Control and Prevention (CDC). These included 26 laboratory reference virus strains and field isolates and 265 geographically (U.S., Central and South America and Africa) and compositionally diverse specimens [nasopharyngeal and oropharyngeal swabs (223), nasal washes and aspirates (21), sputum (1), lung autopsy tissue (1) and unidentified (19)] collected from children and adults with acute respiratory illnesses (ARIs) acquired between 2008 and 2011 and previously testing positive for respiratory viruses by the in-house singleplex assays.
cord-277186-sj8ngpk8	2005	Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The monoclonal antibodies were characterized by SARS CoV-infected Vero cells and nucleocapsid-spike fusion protein-based IFA, Western blot, and N195 proteinbased ELISA. The isotype of the promising monoclonal antibody, designated as S-A5D5, was determined and was further applied to develop a specific and sensitive antigen capture ELISA for the detection of SARS CoV. The specific reactivity of the MAb S-A5D5 with purified N195 protein (Fig. 3A ) was identical to that of the human SARS positive serum (Fig. 3B) , while no reaction was observed when non-antibody secreting hybridoma was tested (Fig. 3C) . Therefore, this antigen capture ELISA, based on MAb to N protein, might provide a more sensitive method for early detection of SARS CoV infection.
cord-277804-ujabzic4	2016	
cord-278176-o9glkhyv	2004	The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The 3′-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region. It was demonstrated that the RT-PCR assay with 91% amplification efficiency could be used for consistent detect ion of the SARS-CoV viral RNA extracted from samples containing as little as 0.005 pfu per reaction with an anticipated C T value of 40 cycles (data not shown). It was demonstrated in this study that the cloned pHCV1 plasmid could be used to replace viral cDNA as a stable and rational SARS-CoV copy number standard for the SARS-CoV RT-PCR assay. Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays
cord-278377-jgq3dz3u	2019	METHODS: Two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (FilmArray Respiratory Panel, Clart Pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. Meanwhile, important questions concerning these ''new'' expensive rapid molecular techniques remain unanswered, such as their cost-effectiveness in terms of patient''s management, or the clinical significance of detecting nucleic acids of micro-organisms that could be non-infectious at the time the sample is collected. The objective of this work was to compare the performances of antigen detection and cell cultures techniques routinely used since years for the diagnosis of respiratory viral infections in the setting of a tertiary care hospital to those of newer molecular techniques (Clart Pneumovir, Genomica, Coslada, Spain and FilmArray Respiratory Panel, Biofire, Biomérieux, Marcy L''Etoile, France). False negative results with molecular techniques were significantly more frequent in samples with codetections compared to those with only one pathogen: 12% vs 3% for the FilmArray test (p = 0.034) and 76% vs 11% for the Clart Pneumovir test (p < 0.001).
cord-279541-rjp2d1u9	2006	Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. The nucleotide sequences (N = 246) of the neuraminidase gene of influenza A virus (H5N1) were taken from the Genbank database going back as far as 2003-2006 and hence, comprising entries isolated from various species, such as avian, cats, dog, tigers, swine and humans, including DQ250165, the sequence of one Vietnamese Oseltamivir-resistant patient (A/Vietnam/CL2009/2005(H5N1)). In conclusion, real-time PCR using two labeled TaqMan probes provides a highly specific and sensitive method to detect the amino acid alteration at position 274 of the influenza A subtype H5N1 neuraminidase gene causing oseltamivir resistance.
cord-279903-z0wf1wli	2014	
cord-281162-2pu7x5rj	2019	The aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with ALRTIs. Methods: The cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (RSV), human metapneumovirus (HMPV), influenza virus A and B (IFV-A and B), parainfluenzavirus 1, 2, 3 and 4 (PIV 1, 2, 3 and 4), human rhinoviruses (HRV), human enterovirus (HEV), human coronaviruses (HCoV) 229E and OC43, human bocavirus (HBoV) and human adenovirus (HAdV) in hospitalized children with ALRTIs, at Hospital Serdang, Malaysia, from June 16 to December 21, 2009.
cord-281174-3c1vue0y	2003	
cord-281999-jc4ckqy7	2008	The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. In the present study, attempts were made to explore the role of proteasome inhibition in ARV infectivity and the mechanisms involved in the proteasome inhibitor suppression of ARV replication and apoptosis induction in cultured cells. Using the proteasome inhibitor MG132 to inhibit the cellular proteasome pathway, it was found that MG132 could reduce ARV-induced apoptosis, cytopathic effect (CPE), virus titer, and protein expression. The results indicated that the expression of A, C, and NS was reduced significantly in BHK-21cells either pre-incubated 30 min before infection or 2 h.p.i. with MG132 (Fig. 2D) , suggesting that the ubiquitin-proteasome system is not involved in virus internalization. In conclusion, it was shown that proteasome inhibitor reduces ARV replication through inhibition of viral RNA transcription and protein synthesis, thus preventing ARV-induced apoptosis.
cord-284644-9k2oox64	2017	Optimized RT-LAMP assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step RT-PCR and real-time RT-PCR. CONCLUSIONS: RT-LAMP assay is rapid, sensitive, specific and cost effective method for detection of influenza A viruses than conventional one-step RT-PCR and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries. The objectives of the current study were to (1) optimize RT-LAMP assay for detection of influenza A viruses and their subtypes (H1N1, H3N2 and pdm09/H1N1); (2) determine sensitivity and specificity of RT-LAMP assay; (3) clinical evaluation of RT-LAMP assay and conventional one-step RT-PCR in comparison to WHO recommended rRT-PCR taken as standard. Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus
cord-286117-m1rlmlun	2020	To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. The Celigo Image Cytometer has demonstrated automated enumeration of viral plaques, foci, and individual virus-infected cells in a 96-well microplates using bright field or fluorescence imaging in less than 5 or 10 min, respectively, which can significantly reduce the time required for counting and analysis as well as eliminate operator-to-operator variation (Ramos et al., 2019; Rosen et al., 2019; Viedma and Pickett, 2018) . Development of the image cytometry-based high-throughput FCoV infection detection method involved the optimization of key variables: host cell seeding density, fluorescent labeling, assay buffer, microplate surface coating, virus concentration, and incubation time. After the optimization of each parameter for the high-throughput virus-infected cell counting method, the image cytometer performed the neutralization assay at approximately 15 min per 96-well plate for simultaneously acquiring and analyzing whole well images in bright field and fluorescence, equivalent to ~9 sec per sample.
cord-286360-wrrqb387	1999	A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), trasmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. Recently a nested polymerase chain reaction (n-PCR) assay was developed for detection of feline infectious peritonitis virus (FIPV), a coronavirus closely related to CCV, in clinical specimens (Gamble et al., 1997) . PCR carried out with CCV1 and CCV2 primers specific for the target sequence 337 -746 of M gene revealed high sensitivity; tests performed on corresponding viral dilutions, which also were inoculated into cell cultures, gave positive results to the 10 − 4 dilution (approximately 10 -50 TCID 50 of virus).
cord-286451-ujo72w06	2012	title: The development of a multiplex real-time PCR for the detection of herpes simplex virus 1 and 2, varizella zoster virus, adenovirus and Chlamydia trachomatis from eye swabs A multiplex real-time PCR assay for rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and Chlamydia trachomatis (C. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. A multiplex real-time PCR assay for the rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and C. This paper describes the development and validation of a multiplex real-time PCR assay, which will allow rapid and simultaneous detection of HSV1/2, VZV, adenovirus and C.
cord-288701-nx9fg4yn	2015	The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. To overcome these limitations, we have developed a multiplex real-time RT-PCR assay for simultaneous detection of the different species of bovine pestiviruses, including the emerging HoBi-like group, allowing a rapid, sensitive and specific diagnosis of pestivirus infection and characterisation of the viral species.
cord-289676-tjy7f9rk	2009	
cord-290831-45cu8alm	1999	Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. French and Roy (1990) constructed a dual-recombinant baculovirus containing genes that encode VP3 and VP7 and their expression in insect cell culture resulted in the synthesis of large quantities of VP3 and VP7 and the concomitant formation of core-like particles (CLP) that lack RNA. Ovine polyclonal antiserum was raised to core particles of BTV-1 isolated from virus-infected cells and purified by equilibrium density centrifugation in cesium chloride (personal communication, B.T. Eaton). The number of crude CLP per negative film was determined by the ISEM method and the concentration of particles calculated by multiplying the number of CLP by the capture efficiency. Serial dilutions of purified CLP were made in uninfected Sf9 cell lysates and 5 ml aliquots of each dilution added to grids coated with a 1:100 dilution of antibody 20E9.
cord-292643-n6xp5mlz	2013	
cord-292831-oihcay6w	2013	The purpose of this study was to develop a one step mRT-PCR that could detect respiratory viruses including influenza A viruses, H1N1pdm09, seasonal H1N1, H3N2, influenza B viruses, respiratory syncytial virus (RSV) A and B, human metapneumovirus (HMPV), parainfluenza viruses (PIV) 1, 2, 3, 4, rhinovirus, enterovirus, corona viruses OC43, 229E, NL63 and HKU1 in three sets in human clinical samples and to compare it with rRT-PCR. The specificity of the multiplex PCR assay was evaluated by cross reaction tests with known viral isolates and different panels of sequence confirmed known clinical respiratory samples as reference material. Specificity of the mRT-PCR assay was evaluated by cross reaction tests against known respiratory virus isolates/positive samples and a WHO QA/QC panel for influenza viruses showed no cross reactivity amongst different viruses. For validation of set 1 of the multiplex assay, 114 respiratory clinical specimens previously positive for influenza viruses by rRT-PCR were used and results were 100% concordant.
cord-293651-96cmduez	2006	
cord-294454-uzfsv2df	2005	Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1–4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses. The aim of this study was to develop rapid, sensitive and specific molecular methods for the detection of a large panel of respiratory RNA viruses that are more powerful than Co-infections hRV + PIV-3 1 hRV + PIV-1 1 hRV + PIV-1 + hMPV 1 hRV + hRSV + hMPV 1 hRV + influenza A virus 1 hRV + hRSV 2 Total no. Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay
cord-295316-ccdj7137	2001	
cord-295401-3p6q92x4	2003	
cord-295491-zlah6u5s	2018	The aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect feline Coronavirus (FCoV) in body cavity effusions of cats with and without FIP, in order to minimize the time from sampling to obtaining results. The aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription LAMP (RT-LAMP) to detect FCoV in body cavity effusions of cats with and without FIP, and to minimize the time from sampling to obtaining results. The FIP group (n = 34) included cats with a definitive diagnosis of FIP by one or more methods: All effusions of cats with FIP tested positive for FCoV by RT-PCR by a commercial laboratory, and in 26/34 samples putative disease-causing mutations could be detected.
cord-297160-tqw9vx2b	2013	After the incubation period of seven days standard practice is for the embryos to be taken from each egg and examined visually for IB specific lesions; these readings are used to determine an end-point in viral titrations. In order to determine end-points in the titration for each of the two strains, we collected the allantoic fluids from each egg after the incubation period and tested these for the presence of IB H120 and IB D274 by a strain specific reverse phase PCR. We used PCR for detection of two different IB virus strains in the allantoic fluids from all the individual eggs in a titration of a live IB combination vaccine. Based on the results of the PCR we determined whether an allantoic fluid was positive for one or both of the viruses or not, and as such we were able to define a separate endpoint in the live virus titration for each of the two strains.
cord-298922-k568hlf4	2015	
cord-299585-fkg8d6ym	2019	
cord-301355-9lswjro2	2015	
cord-301430-gzou8b9k	2004	
cord-301974-4wn40ivq	2004	
cord-302024-zz7mt6be	2004	
cord-302663-gb2vgs97	2006	Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The development of a loop-mediated isothermal amplification (LAMP) assay for detection of white spot disease virus (WSDV) DNA was described by Kono et al. Ten-fold serial dilutions (10 −1 to 10 −8 diluted) of RNA extracted from YHV-infected shrimp was used as a template for RT-LAMP according to determined conditions. In order to determine the sensitivity of detection limit, RT-LAMP and nested RT-PCR were carried out using various concentrations (10 −1 to 10 −8 dilution) of RNA extracted from YHV-infected shrimp as template.
cord-302829-1o1jo8uk	2008	A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). DNA was extracted from blood, lymph nodes, lung, liver, kidney, heart and spleen samples taken from PCV2-infected and healthy pigs, using a DNeasy Tissue Kit (Qiagen) according to the manufacturer''s instructions. In order to evaluate the optimal tissues for viral detection and to compare the sensitivity of PCV2 detection by LAMP and PCR, DNAs from tissue samples of blood, heart, lung, liver, kidney, lymph nodes and spleen from PCV2-infected pigs were extracted and subjected to LAMP and PCR. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction
cord-305399-98sqovwb	2019	
cord-305640-tgowzrqo	2005	A capture enzyme-enhanced chemiluminescence immunoassay (ECLIA) based on three specific monoclonal antibodies to detect the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in the serial serum samples from SARS patients was developed. The N protein is an extensively phosphorylated, highly basic protein, which interacts with viral RNA and makes up the viral core and nucleocapsid (Lai, 2003 the diagnosis of SARS depends basically upon detecting SARS-CoV RNA by RT-PCR and/or testing specific antibodies directed against SARS-CoV by assays based on cultured virus or recombinant viral antigens. In the present study, a capture ECLIA was developed based on three monoclonal antibodies directed against the N protein of SARS-CoV, and the N protein in the longitudinal serum samples from the SARS patients were detected with this method. The detection of the N protein of SARS-CoV in serum samples by ECLIA appears to be superior to the detection of the viral RNA by RT-PCR in rapid diagnosis of SARS patients.
cord-307304-irji8owi	2004	To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs. Avian infectious bronchitis virus (IBV), a group three member of the genus Coronavirus (order Nidovirales, family Coronaviridae), is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct (Cavanagh, 2001; Cavanagh and Naqi, 2003; Cook et al., 2001) . In an alternative strategy infectious IBV was recovered following transfection of restricted vaccinia virus DNA, containing the IBV full-length cDNA, into cells infected with recombinant fowlpox virus expressing T7 RNA polymerase (Casais et al., 2001) .
cord-308338-lhe51ws7	2011	
cord-310771-tnwfp1je	2005	A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = 36). For the development of one-step real-time RT-PCR for four endogenous reference RNAs (HPRT, UBE2D2, PPIA, and HMBS) appropriate target regions were selected and the assay conditions were optimised for amplification efficiency. One-step real-time RT-PCR assays for PRRSV-1 and -2 RNA allowed quantitation with optimal efficiency (Fig. 1a ; standard curves for two additional viremic pigs infected with PRRSV-1 (data not shown)) as achieved for endogenous and Fig. 1 .
cord-311410-lgqup9ug	2006	
cord-311639-zij2wbzs	2012	This study was performed to evaluate the analytical and clinical performance of a newly developed rapid ICG test (SD Bioline Norovirus test) for detecting human norovirus genogroups GI and GII in stool specimens. In samples with negative ICG and positive real-time PCR results, 200 L of fecal suspension was mixed with 200 L diluent (1:1 dilution) instead of 1 mL diluent, and the test was repeated. In this study, therefore, in the case of samples with negative ICG and positive real-time PCR results, 200 L of fecal suspension was mixed with 200 L diluent instead of 1 mL diluent (total dilution titer was 1:10-1:20 dilution, which is similar to that of the original procedure of this assay using stool), and the test was repeated. Evaluation of rapid immunochromatography test for the detection of norovirus infection: comparison with ELISA and real time quantitative reverse transcription PCR assays
cord-311801-m2otfdjw	2020	
cord-312456-6lxc2rj2	2016	
cord-313271-2e8vjtop	2006	
cord-313541-fpqwzf9k	2020	
cord-317307-q5mgue5z	2009	For evaluating IFA sensitivity, 10-fold dilutions (ranging from 10 2 to 10 −2 TCID 50 /200 l) of titrated virus were obtained and different variables, such as days of incubation (2-4 days) and primary monoclonal antibody dilutions (MAb; 1:40-1:80-1:160 in albumin supplemented with PBS 1%), were examined. Different concentrations of TCID 50 (ranged from 10 2 to 10 −5 /reaction) were amplified by the RT Real Time Qt-PCRs in order to compare sensitivity and specificity of the two diagnostic approaches. For the RT Real Time Qt-PCRs, the optimal parameters in obtaining the lowest detection limit with a high specificity resulted in the following concentrations: both primers 0.9 mM and probe 0.25 mM for HPIV-1 and HPIV-2; forward primer 1 mM and reverse 0.9 mM, and probe 0.25 mM for HPIV-3 amplification. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4
cord-317462-nvrl0vyi	2016	To gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (TGEV) in porcine intestinal epithelial cells (IECs), this study established a cell model of IECs infected with the Chongqing (CQ) strain of TGEV. The results also showed that from 0 to 12 h after TGEV infection of porcine IECs, the intracellular viral RNA content did not change significantly. Thus, we developed an in vitro model based on porcine intestinal epithelial cells (IEC) infected with TGEV, and used transmission electron microscopy (TEM), indirect immunofluorescence assay (IFA) and real-time fluorescence quantitative PCR (FQ-PCR) to investigate the infection mechanism of TGEV. TGEV CQ strain was used to infect porcine IECs, and the supernatant and cells were collected at different time points for FQ-PCR. The one-step growth curve of TGEV in porcine IECs showed that the amount of viral RNA did not change significantly from 0 to 12 h post-infection, whereas Dai et al.
cord-319392-zg7gkf0j	2011	
cord-320492-1xyjrjpf	2015	
cord-321886-0b3ocoh9	2010	title: Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). Because of its sensitivity, specificity, and simplicity, the LAMP assay could be a useful method for early and rapid differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus. Based on PRV-gG-LAMP, 26 of the 70 piglets were infected with wild-type PRV and may or may not have been vaccinated. Many different PCR assays can differentiate between the wild-type PRV and gene-deleted virus vaccines (Liu et al., 2007) , but LAMP is easier to perform and provides more rapid results. (2008) described a LAMP system for PRV detection but that LAMP system could not differentiate between swine infected with wild-type PRV and swine vaccinated with PRV gE-deleted.
cord-322143-hkh1grys	2017	For instance, understanding the persistence of human enteric viruses on inanimate fomite surfaces in relation to various environmental conditions could provide insight on ways to limit and prevent virus transmission and subsequent outbreaks. Overall, the higher the inoculum level for all enteric viruses, the higher the mean recovery rate regardless of the variability among methods, PA = plaque assay; PBS = phosphate buffered saline; PBST = PBS + 0.02% Tween 80; PCRU = polymerase chain reaction units; PE = polyethylene; PF = porous formic; PFU = plaque forming units; RH = relative humidity; RB = rubberized surface; RT-qPCR = reverse transcription quantitative PCR; RT = room temperature; SS = stainless steel. Additionally, some studies found other tools and methods such as biowipes and cell scraper-aspiration methods to be potentially more efficient for enteric virus recovery from surfaces in comparison to cotton and/or polyester swabs.
cord-322234-1zyy536y	2009	
cord-322410-k23engcx	2017	title: New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples. The specificity of RT-qPCR primer set was evaluated by examining different avian respiratory viruses circulating in poultry in Egypt including AIV H5N1, H9N2 and NDV as well as different coronaviruses including a panel of IBV reference strains as listed in the materials section. Within HVR 1, 2 sequences, however, the existence of two distinct Table 3 RT-qPCRs reveal frequent co-infections in Egyptian poultry samples with avian influenza (AIV), Newcastle Disease (NDV) and infectious bronchitis viruses (IBV).
cord-323072-4rsgeag7	2004	Since the outbreak of SARS in 2003, several laboratory diagnostic methods have been established, including real-time RT-PCR assay, whole-virus-based immunofluorescence assay (IFA), recombinant protein-based enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests, antigencapturing enzyme-linked immunosorbent assay, and Western blot (WB) assay. To test whether the recombinant M protein is effective as an ELISA antigen for detecting SARS-CoV patient serum, the sera from four healthy people and four SARS patients were used. Detection results of eight human sera by ELISA using purified recombinant M protein as antigen.# 1-4: sera from four healthy people, respectively, # 5-8: sera from four SARS patients, respectively. The results were in complete accordance with those of other assays, thus indicating that the recombinant M protein may be useful as an ELISA antigen for detecting specific antibodies to SARS-CoV in human sera. Recombinant protein-based enzyme-linked imunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients
cord-324213-3uqlimov	2009	To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. To determine the limit of detection (LOD) of the H275Y and N1 control RT-PCR assay, serial ten-fold dilutions of nucleic acid from an oseltamivir-sensitive clinical influenza A/Brisbane/57/2007 H1N1 isolate were tested. This study evaluated the use of a novel H275Y RT-PCR assay for detection of H275Y-positive isolates in comparison to Sanger sequencing and pyrosequencing, and found that while all three methods may have a role in influenza diagnostic testing, the H275Y RT-PCR assay is a rapid and effective test for the detection of oseltamivir resistance through mutation at residue 275.
cord-326225-crtpzad7	2014	This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. There is a wealth of information in these isolates, but up till now, it has been time consuming and expensive to sequence these viral genomes, often requiring sets of strain-specific primers for PCR amplification and sequencing. These primers were developed so that the 20 base known sequence was used for PCR amplification of the library as well as served as a barcode for identifying each viral library following pooling and sequencing. This virus, a BVDV 1b strain isolated from alpaca (GenBank accession JX297520.1; Table 2 , library 3, barcode 10), was assembled from Ion Torrent data and was found to have only 1 base difference from the sequence determined earlier (data not shown). One virus, library 1, barcode 9, had only 658 viral sequence reads but 94.4% of the genome was assembled.
cord-328961-waxtb759	2002	Comparative sequence analysis of the PCR products of the M gene and fragments of the pol1a and pol1b genes of canine coronavirus (CCoV) have demonstrated that two separate clusters of CCoV are present in dogs. The sequence analysis of the PCR products of the M and S genes carried out on the faecal samples from the two pups confirmed that the FCoV-like CCoV had caused the disease (personal observations). On the basis of these preliminary results, a PCR assay was developed to detect and identify the FCoV-like CCoV strains from faecal samples of infected dogs. In a previous study, similar nucleotide substitutions in the binding site of the internal primer CCoV3 used for the n-PCR were demonstrated in the sequence analysis of the PCR products from five faecal samples of pups with diarrhoea.
cord-331509-p19dg1jw	2020	title: Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. For a rapid, accurate and reliable diagnosis of PED in the veterinary laboratory, a method for the detection of PEDV viral RNA has been developed and more importantly validated according to the "Association Francaise de NORmalisation" (AFNOR) French NF U47-600 norm entitled "requirement and recommendation for the implementation, development and validation of PCR in animal health" (AFNOR, 2015a; AFNOR, 2015b) . This method should help harmonize detection and quantification of viral RNA from PEDV belonging to both S-non-INDEL and S-INDEL strains in both field and experimental settings.
cord-332522-adul9nzf	2004	In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages. To compare the sensitivities of these 12 sets of nested primers, serial 10-fold di-lution genome cDNA of BJ01 that reverse transcribed with random primer was used as the template to carry out the nested PCR.
cord-336639-jaue41mv	2004	A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The reason for this discrepancy became clear when the biological and genetic properties of FECV and FIPV isolates had been studied (Addie and Jarrett, 1992; Hohdatsu et al., 1992; Horzinek and Osterhaus, 1979) : the avirulent FCoV strains causing inconspicuous infections are responsible for the high seroprevalence; in cats experiencing some immunosuppressive event, expansion of the quasispecies cloud and mutations in the FECV genome lead to virulent variants that induce FIP (Vennema et al., 1998) . Detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis
cord-343136-kftffes0	2020	A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. Limit of detection of the LAMP assay was evaluated by using a nasopharyngeal (NP) swab sample infected with SARS-CoV-2 for which the viral load was quantified using digital droplet PCR (see Supplementary Methods). Twenty four replicates from a serial dilution containing 25-50 copies of SARS-CoV-2 which equates to 1X LOD (patient sample NP swab in VTM viral load confirmed by digital droplet PCR) per reaction were tested using dual-target RT-LAMP (Table 3) . The dual-target RT-LAMP test for SARS-CoV-2 developed in this study has comparable analytical sensitivity and specificity, limit of detection, precision, and achieved excellent agreement compared to the reference RT-PCR methods used internationally.
cord-343441-z849jvq5	2013	In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The analytic sensitivity of the duplex TaqMan rRT-PCR assay was compared with the WHO TaqMan assay and a commercial single H7N9 rRT-PCR kit (bioPerfectus technologies, Taizhou, China) with a 10-fold dilution series of a nasopharyngeal aspirate (NPA) from a patient infected with the H7N9 virus (approximately 4.8 × 10 6 copies of the viral genome/mL). To determine the actual detection limit (number of copies per reaction) of the duplex TaqMan rRT-PCR assay, in vitro RNA transcripts of HA and NA genes from the H7N9 virus were prepared with T7 RNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian, China) according to the manufacturer''s instructions using influenza A/Nanjing/1/2013 (H7N9) RNA as a template.
cord-350753-qbm145tr	2020	Using 75 sera from patients tested positive or negative by SARS-CoV-2 PCR, we investigated the sensitivity and specificity of the Liaison SARS-CoV-2 S1/S2 IgG assay (DiaSorin), the Elecsys Anti-SARS-CoV-2 assay (Roche), and the ID Screen SARS-CoV-2-N IgG indirect kit (IDVet). We and others have published results of the assessment of the first commercially available serological assays, such as the Anti SARS-CoV-2 ELISA (IgG) from Euroimmun (Krüttgen et al., 2020; Okba et al., 2020) . We therefore compared these three new assays with respect to their sensitivity and specificity to detect SARS-CoV-2 specific antibodies using a collection of serum samples employed previously for the analysis of four other assays. Our comparative approach to test in total seven different SARS-CoV-2 antibody assays with an identical collection of serum samples allowed for the first time the direct comparison of performance indicators of such a large number of automated assays.