key: cord-0939092-0gtrhe89
authors: Erickson, A.; Fisher, M.; Furukawa‐Stoffer, T.; Ambagala, A.; Hodko, D.; Pasick, J.; King, D. P.; Nfon, C.; Ortega Polo, R.; Lung, O.
title: A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine
date: 2017-11-30
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.12749
sha: 9c6206c953a6297141e710dfa1971b68b2c6a0c9
doc_id: 939092
cord_uid: 0gtrhe89

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour‐intensive and time‐consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)‐PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot‐and‐mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user‐friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post‐infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT‐PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.

The swine industry is a major part of the global livestock industry and an important part of the food supply chain. The global pig industry produced approximately 963 million pigs and 109 million metric tons of pork in 2011 (http://faostat.fao.org). Infectious diseases such as foot-and-mouth disease (FMD), classical swine fever (CSF) and

African swine fever (ASF) are highly contagious viral diseases reportable to the OIE (World Organization for Animal Health) that can have a severe economic impact on affected areas due to production losses and the impact on the international trade of animals and animal products with disease-free countries. For example, the 2001 FMD outbreak in the United Kingdom had an estimated cost of $13 billion US (Thompson et al., 2002) . The direct costs of the 1997-1998 CSF epizootic in the Netherlands, excluding loss of exports, amounted to $2 billion US and the slaughter of approximately 10 million pigs (Terpstra & de Smit, 2000) .

FMD affects over 70 species of cloven-hoofed animals including cattle, pigs, sheep, goats and wild ruminants causing vesicular lesions on the mouth and hoof (Hedger, Condy, & Gradwell, 1980) . The disease is endemic in many parts of South America, Asia and Africa.

The aetiological agent is the FMD virus (FMDV, Picornaviridae, Aphthovirus). Non-FMD viruses that can cause clinically indistinguishable vesicular lesions in swine include swine vesicular disease virus (SVDV, Picornaviridae, Enterovirus) and vesicular exanthema of swine virus (VESV, Caliciviridae), a virus derived from feeding pigs seal meat contaminated with San Miguel sea lion virus (SMSV; Zimmerman, Karriker, Ramirez, Schwartz, & Stevenson, 2012) . CSF virus (CSFV, Flaviviridae) causes haemorrhagic disease in pigs (Giammarioli, Pellegrini, Casciari, & Mia, 2008) and lesions in less severe cases (Zimmerman et al., 2012) . African swine fever virus (ASFV, Asfarviridae) also causes haemorrhagic fever (Costard et al., 2009; Giammarioli et al., 2008 ) that is clinically indistinguishable from infection with CSFV.

For the laboratory diagnosis of vesicular diseases, the OIE currently recommends methods such as virus isolation, antigen capture enzyme-linked immunosorbent assay (ELISA) and PCR, including realtime PCR. Virus isolation is often considered the gold standard, but ELISA and real-time reverse transcriptase (RT)-PCR are faster and thus more suitable for use as screening tests. Conventional and real-time RT-PCR assays have been developed for CSFV (Wernike, Hoffmann, & Beer, 2013) , ASFV (Fern andez-Pinero et al., 2013) and FMDV (Hole, Clavijo, & Pineda, 2006; King et al., 2006; Reid et al., 2009 ) and its differentials that include vesicular stomatitis virus (VSV; Hole et al., 2006; Rasmussen, Uttenthal, & Ag€ uero, 2006) , SVDV (Fern andez et al., 2008; N uñez et al., 1998) , and VESV (Reid et al., 2007) . Userfriendly reverse transcription insulated isothermal PCR (RT-iiPCR) assays performed on compact, field-deployable instruments with automatic display of "+" or "À" results have also been described for CSFV (Lung et al., 2015) and FMDV . Real-time multiplex PCR assays have been reported for the detection of FMDV and CSFV (Shi et al., 2016; Wernike et al., 2013) and other targets (Haines, Hofmann, King, Drew, & Crooke, 2013; Li et al., 2013; Rasmussen et al., 2006; Wernike et al., 2012) . Although RT-iiPCR and real-time RT-PCR are rapid and highly sensitive, separate single-target reactions are normally performed by the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease when testing for the presence of foreign animal disease pathogens. Thus, a userfriendly, multiplex diagnostic test for differential diagnosis of highconsequence foreign animal diseases could be a useful tool.

In addition to the major vesicular diseases of swine, CSFV and ASFV, two viruses indigenous to North America; porcine circovirus type 2 (PCV2, Circoviridae) and porcine respiratory and reproductive syndrome virus (PRRSV, Arteriviridae), which are responsible for most of the production losses to the North American pig industry (Nicholson et al., 2011; Zimmerman et al., 2012) , were also tested with available resources to evaluate the potential of this technology for testing of indigenous diseases. Depending on the strain of the virus and the immune status of the host, there is considerable variation in clinical signs of PRRS. Typically, PRRSV infection causes mild to severe respiratory disease in newborn and growing pigs and reproductive failure in pregnant sows (Lunney et al., 2016) and can also cause apoptosis in organs such as the lungs, testes, lymph nodes and thymus (Karniychuk et al., 2011) . PCV2 infection can lead to lymphoid depletion and immunosuppression in pigs and is the primary causative agent of porcine circovirus-associated disease (PCVAD). Infections can also cause wasting and increased mortality, as well as lesions in the lymphatic area (Meng, 2013) .

Microarrays can accommodate multiple probes for each target that can provide redundancy and broader coverage of viral variants when compared with single probe assays. The development and evaluation of traditional microarrays for subtyping and multiplex detection of viruses that affect livestock have been previously described (Ban er et al., 2007; Hindson et al., 2008; Jack et al., 2009; Lenhoff et al., 2008; Lung et al., 2011 Lung et al., , 2016 . The assay described by Ban er et al. (2007) detected FMDV, SVDV, VSV, and differentiated between the two serotypes of VSV. Lung et al. (2011) described an assay that detected four vesicular disease viruses (FMDV, VSV, SVDV and VESV) and differentiated between the seven serotypes of FMDV (A, O, C, Asia 1, SAT1, SAT2 and SAT3) and the two serotypes of VSV (New Jersey & Indiana). These methods are laborious to perform, utilize conventional slide microarrays that rely on slower passive hybridization and require multiple pieces of equipment for capture probe printing, hybridization, washing, and slide scanning. In this study, the development and initial validation of an electronic microarray platform that integrates and automates ERICKSON ET AL. | e273 capture probe printing with microarray hybridization, washing, and reporting on a single instrument and allows for onsite updating of probe sequences and custom printing of capture probes by the user for the detection of multiple viruses is described.

Sixty-eight laboratory and field isolates of the seven targeted swine viruses were used in this study (Tables 1, 5 ). The strains tested represent viruses from all seven FMDV serotypes, three CSFV genotypes, and both North American (NA) and European (EU) genotypes of PRRSV that were available for testing at the Canadian Food Inspection Agency (CFIA). Strains of FMDV, SVDV, VESV, CSFV and ASFV were propagated and titered according to previously published methods (Moniwa, Clavijo, Li, Collignon, & Kitching, 2007; Senthilkumaran et al., 2016) . PRRSV RNA and PCV2 DNA were kindly provided by 

The details of the inoculation studies in animals and clinical samples were as previously described (Lung et al., 2011) or will be described 

PCR primer and capture probe sequences were either identified from the literature, then used with or without modifications or were newly designed (Tables 2 and 3 ). Primer and probe design were performed as described previously (Lung et al., 2011 (Lung et al., , 2012 using AlleleID â v. all the unique primer pairs in the multiplex PCR (e.g., the forward primer of FMDV and the reverse primer of ASFV) that target viruses other than the viruses for which the primers were originally designed.

The table was used as the input for the e-PCR program to test the primers in silico. Hits that were returned with less than four mismatches were evaluated with ThermoBLAST's in silico simulations under reaction conditions to predict possible hybridizations based on thermodynamic parameters as well as sequence complementarity (SantaLucia, 2007) . Candidate probes were screened for predicted specificity using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Virus-specific candidate probes that did not show significant matches to the other target viruses (probes exhibiting <25% overall identity and cross-homologous regions of less than eight nucleotides) were then screened on the NanoChip400 microarray (Nexogen) against a reference panel of viruses. All capture probes were modified with 5 0biotinylation to allow attachment to the streptavidin-containing hydrogel of the microarray. All primers and probes were synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA).

Nucleic acid was extracted from vesicular viruses (FMDV, SVDV and VESV) and clinical samples as described previously (Lung et al., 2011) , while all other viruses were extracted using the QIAamp Viral RNA Kit (Deregt et al., 2006) 

All buffers used in the computer-controlled automated electronic microarray assay were from Nexogen, Inc. or prepared in-house from chemicals purchased from Sigma-Aldrich (Oakville, ON, Canada).

Biotinylated capture probes (250 nM) were prepared in 50 mM Lhistidine/0.05% Proclin â 300 buffer (His/Proclin â buffer, Nexogen

Inc.) and electronically addressed to selected electrodes on the (Sambrook & Russell, 2001) .

Copy number was calculated using the following equation:

concentration ðngÞ Â 6:022  10 23 =mole base pair ðbpÞ Â 1  10 9 ng=g  Molecular Weight bp g=mole After quantification, in vitro transcribed RNA and plasmid DNA were diluted serially 10-fold from neat stock to 10 À12 in T A B L E 2 PCR primers used in this study.

Genomic region Primer Sequence (5 0 -3 0 ) 

The multiplex RT-PCR utilized 22 primers to amplify selected genomic regions of the seven target viruses ( Table 2 ). The RT-PCR generated amplicons of the expected size for all 68 isolates of the seven target viruses (23 FMDV, 12 SVDV, 10 CSFV, one VESV, three ASFV, one PCV2 and 18 PRRSV; Figures 1a and 3a) . The specificity of the amplification was evaluated using a total of 22 samples, including 11 oral clinical materials from healthy pigs, and 11 non-target swine bacteria and viruses associated with livestock. Nucleic acid extracted from non-target pathogens and clinical material from healthy animals either did not generate detectable RT-PCR products or generated weak, non-specific products in the absence of templates from target viruses (Figure 1b) . The nonspecific amplifications were reduced substantially when the same samples were spiked with PRRSV RNA as an exogenous control (Figure 1c) .

Virus/Genomic region Probe name Sequence (5 0 biotinylation a -3 0 ) Reference A  AAG TTG GCN GGA GAC GTB GAG TCC AAC CC  This study   FMD Common B  AAC TTY GAC CTG TTA AAG TTG GCB GGA GAC GTT GAG TC  This study   FMD Common C  AAC TTC GAC CTG TTA AAG TTG GCY GGA GAC  GTT GAG TCC Probes modified with 5 0 biotinylation for binding to streptavidin pad on NanoChip 400 microarray (Takahashi, Norman, Mather, & Patterson, 2008 

Nucleic acid from 10-fold serial dilutions of transcribed RNA of known copy number were amplified by single-plex RT-PCR, a duplex RT-PCR consisting of primers for PRRSV and PCV2, a five-plex RT-PCR for viruses exotic to Canada (FMDV, SVDV, VESV, CSFV and ASFV) or the seven-plex RT-PCR. The RT-PCR amplicons were then tested on the microarray, to determine the limit of detection of the RT-PCRs and microarray for each virus. The assay showed similar efficiencies for the single-plex, duplex and five-plex RT-PCRs and microarray for SVDV, VESV, CSFV, ASFV, PCV2 and PRRSV, but the PCR had higher sensitivity for the smaller amplicons: ASFV, PCV2

and PRRSV (Table 4 ). The seven-plex RT-PCR showed the best analytical sensitivity with ASFV, PCV2 and PRRSV at a range between 1 copy in PCR and 10 copies on the electronic microarray, followed by SVDV, CSFV and VESV at around 10 and 100 copies in PCR and microarray, respectively. The detection limit was lowest for FMDV at hundreds of copies for both seven-plex and five-plex PCRs, and one (single-plex, five-plex) or two (seven-plex) orders of magnitude lower on the microarray (Table 4 ).

Extracted nucleic acid from clinical samples was amplified using the seven-plex multiplex assay. FMDV nucleic acid was detected in serum samples as early as 1 day post-infection (dpi). All available targets were detected from nasal, oral, serum or whole blood with detection ranging from 1 dpi for SVDV, 4 dpi for ASFV and 5 dpi for CSFV (Table 5) . PRRSV was detected in positive field serum samples from swine (n = 10, Table 5, Figure 3 ). All samples from oral and/or nasal swab material that were spiked with VESV, PRRSV and PCV2 (n = 8) produced amplicons of the expected size after the multiplex PCR and the viruses were accurately detected by the microarray (data not shown).

In addition to samples taken prior to experimental infection at 0 dpi, nucleic acid extracted from oral swabs from 11 healthy swine did not produce any amplicons or produced weak detectable bands on agarose gels after the multiplex RT-PCR and no reactivity above background was observed subsequently on the microarray, demonstrating 100% specificity for these samples ( Figure 2b ).

This study describes the development and initial laboratory evaluation of a novel user-friendly microarray primarily designed for the detection and differentiation of FMDV, SVDV, VESV, CSFV, and = 8) , and PRRSV-positive field samples (n = 10). All capture probes were highly specific and showed no cross-reactivity with heterologous viruses, non-target bacteria and viruses, or samples from healthy animals. PRRSV was originally detected in North America and Europe almost simultaneously (Christianson & Joo, 1994; Lunney et al., 2016) . The two genotypes were genetically diverse with about 55%-70% nucleotide identity (Lunney et al., 2016 (Haines et al., 2013; Li et al., 2013; Rasmussen et al., 2006; Shi et al., 2016; Wernike et al., 2012 Wernike et al., , 2013 , the diagnosis of FMDV, SVDV, CSFV and ASFV still requires the performance of multiple single tests. Automated multiplex assays can potentially reduce labour cost and handling time. The highest analytical sensitivities observed for the 7-plex RT-PCR were as follows: PRRSV, PCV2 and ASFV which have the smallest amplicons (379-537 bp). Although the microarray assay successfully detected FMDV in dpi 1 samples from experimentally infected animals, the multiplex RT-PCR was less efficient for amplification of FMDV, which had the largest amplicon size in the assay (971 bp). This is most likely due to reduced PCR efficiencies for large amplicons (Cheng, Fockler, Barnes, & Higuchi, 1994; Huang, Arnheim, & Goodman, 1992) . The FMDV primers used in this study were specifically designed to amplify the highly variable Isolate F I G U R E 3 Multiplex RT-PCR amplicons visualized using QIAxcel System (a) and heat map depicting the reactivity of 10 genetically diverse Canadian PRRSV-positive serum samples from diagnostic submissions against 27 virus-specific capture probes and one nonspecific binding probe (NSBP) (b). The panel of samples includes the following: #2 (17-001377-0001_1-57-1), #7 (17-007044-2208_1-3-2), #11 (17-010599-0006_1-30-2), #20 (17-019861-0006_1-1-1), #21 (17-020077-0002_1-8-4), #22 (17-020084-0001_1-3-2),-#23 (17-020347-0011_1-8-2), #33 (17-035-986_1-4-1), #36 (17-039858-0022_2-5-2), #43 (17-052259-0023_1-57-1). A positive signal in red represents a positive-to-negative ratio (P/N) of >2, while negative results in black represent any P/N ≤ 2 capture probes, the amplicon or the annealing of the two strands of the amplicons which could prevent efficient hybridization between the capture probes and the amplicon under the experimental conditions. The use of asymmetric PCR to generate single-stranded products, increased amount of capture probe and/or amplicon may also further increase the sensitivity of the assay. Although not tested in this study due to resource constraints, previously published electronic microarray assays for detection of avian, swine and bovine viruses (Lung et al., 2012 (Lung et al., , 2017 , were able to detect the presence of more than one target. Due to potentially high loads of viruses such as PCV2 in many countries, the splitting of the sevenplex RT-PCR into a duplex RT-PCR for PRRSV and PCV2 and a fiveplex RT-PCR for the other viruses, as described in this study, will improve the sensitivity of the assay for the detection of reportable diseases.

Although an RT step was not required for DNA viruses, previous studies have shown better amplification of the DNA targets when a RT step was included (Lung et al., 2017) . This could be due to the utilization of messenger RNAs as well as genomic DNA as template (Lung et al., 2017) . Further improvements in assay sensitivity and integration of the PCR with the electronic microarray in the new NanoChip400 XL electronic microarray platform could make this novel automated microarray technology a useful tool for multipathogen detection and subtyping.

CRTI) project 09-403TA. The authors acknowledge Dr. Noriko Goji, Mr. Sandor Dudas, and two anonymous reviewers for suggestions

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How to cite this article

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

Canada, Centre for Security Science's Chemical, Biological, Radiological-Nuclear, and Explosives Research and Technology Initiative