key: cord-0005539-vqss7z7m authors: Zhang, Lei; Liu, Ye-bing; Chen, Lei; Wang, Jian-huan; Ning, Yi-bao title: Rapid and sensitive detection of PRRSV by a reverse transcription-loop-mediated isothermal amplification assay date: 2011-08-17 journal: Virol Sin DOI: 10.1007/s12250-011-3185-x sha: 28117bc12436bf62530b21a0c0b22573eddfb0d1 doc_id: 5539 cord_uid: vqss7z7m A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic, prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains. As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR), the RT-LAMP method only used a turbidimeter, exhibited a detection limit corresponding to a 10(−4) dilution of template RNA extracted from 250 μL of 10(5) of the 50% tissue culture infective dose (TCID(50)) of PRRSV-containing cells, and no cross-reactivity was observed with other related viruses including porcine circovirus type 2, swine influenza virus, porcine rotavirus and classical swine fever virus. From forty-two field samples, 33 samples in the RT-LAMP assay was detected positive, whereas three of which were not detected by RT-PCR. Furthermore, in 33 strains of PRRSV, an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages. These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates, especially in developing countries. Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease causing severe economic losses in the swine industry. It is characterized by reproductive failure, respiratory disease and various clinical signs including loss of appetite, fever, dyspnea and mild neurological symptoms [5, 17] . There are two major PRRSV genotypes [10] , referred to as North American and European, and the North American genotype is predominant in China. Previous studies have shown that the non-structural protein 2 (nsp2) coding gene, in particular the central region of this gene, may contribute largely to the genetic variation in PRRSV genomes, while high conservation is exhibited in the N-terminal putative protease domain and the C-terminal predicted transmembrane region [18] . In addition, the Nsp2 protein of PRRSV possesses potential enzymatic function, although this was shown to be highly heterogeneous and variable 253 [15] . Since the first widespread outbreak of PRRS in China in 2006, atypical PRRS, caused by variations in PRRSV strain VR2332, has been reported in more than 10 Chinese provinces and regions [9] . Since the development of loop-mediated isothermal amplification (LAMP) in 2000 by Notomi et al [12] , this novel detection method has been extensively used to detect viruses of a wide range of species from humans to animals [4, 14] . This method offers the advantages of simplicity and sensitivity, which can be performed in one step, in real time. Reverse transcription (RT)-LAMP relies on an auto-cycling strand displacement DNA synthesis reaction performed using the Bst DNA polymerase large fragment under isothermal conditions at a temperature of between 60°C and 65°C, which enables the amplification reaction to be performed in a heating block without the need for precise centralized laboratory facilities [3] . This technique is high specificity because the target sequence is recognized by six independent sequences in the initial stage and by four independent sequences during the later stages of the RT-LAMP reaction. This method generates ascendant turbidity in positive samples, allowing detection by the naked eye or real-time monitoring. Previous reports have indicated that the nsp2 region of the PRRSV genome contains restriction enzyme sites, so in this organism, amplification can be further confirmed by enzyme digestion. Several studies have reported the rapid detection of PRRSV using a RT-LAMP assay [1] . In this study, we developed another RT-LAMP assay to detect prototypic and prevalent North American PRRSV strains that Table 1 . A total of 42 suspected field samples of PRRSV collected from farms in Jiangxi and Guangdong province in 2008. Each specimen was from one piglet or fetus and comprised liver, spleen, lung, lymph node and one or more serum samples. All samples were stored at −80°C prior to use. Total RNA was extracted from clinical samples or cell culture supernatants of PRRSV isolates using an RNA extraction kit (TaKaRa) according to the manufacturer's instructions. The RT-LAMP reaction was carried out in a total reaction volume of 25 μL containing 12. Total RNA was extracted from the collected field samples and analyzed by the RT-LAMP assay. As a control, a conventional RT-PCR, following the methods of Yue et al. [19] , was performed simultaneously. To validate the RT-LAMP results, virus was isolated from 42 clinical samples. Porcine alveolar macrophages were used to isolate the virus. Virus isolation followed the methods of Osorio et al [13] . The viruses isolated from samples were tested as outlined by Gonnie Nodelijk et al [11] . Samples were considered negative after two blind passages. The RT-LAMP assay was standardized using PRRSV strain BJ, isolated from a patient with clinically diagnosed PRRS. The RT-LAMP assay successfully amplified the target sequence of the PRRSV nsp2 region and this was confirmed by agarose gel electrophoresis (Fig. 1) . The amplification products appeared in a ladder-like pattern on the gel due to the formation of a mixture of stem-loop DNAs of various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats in the target sequence in the same strand. In the negative control, no specific amplification products were observed, in accordance with the results collected by the Loopamp real-time turbidimeter (Fig. 2) . The RT-LAMP products were digested with SupI and analyzed by 1.5% agarose gel electrophoresis. For strain VR-2332, the RT-LAMP product was digested into two bands of 160 bp and 120 bp, whereas the RT-LAMP product of the highly pathogenic isolate BJ was not digested by SupI (Fig. 3) . The sensitivity of the RT-LAMP assay for the detection of PRRSV North American strains was determined using 10-fold serial dilutions of virus that had previously been quantified by the TCID 50 assay and comparing these results with those of conventional RT-PCR. The RT-LAMP assay was able to amplify the virus in 60 min, with a detection limit corresponding to a 10 -5 dilution of template RNA (Fig. 4) . On comparing the two assays, RT-LAMP was found to be 100-fold more sensitive than RT-PCR, Following digestion with SpuI, DNA fragments were cloned and sequenced. Sequence analysis indicated over 97% homology between the sequence of the amplified fragment and the nucleotide sequence published in the NCBI database, indicating that the RT-LAMP assay was highly specific. Table 2) . There are three methods commonly used for virus [16] . Serological tests, such as enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays, generally require complicated procedures and expensive antibodies and are therefore not widely applicable, especially for early or pre-clinical diagnoses [2, 7, 8] . Although reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive and specific method, the dependence on specialized equipment limits its use in the field or in basic laboratories [2, 6] . The development of molecular detection methods to simplify the diagnosis procedure is of great importance. RT-LAMP is a one-step assay that does not require a cDNA synthesis step, simplifying the method and decreasing the risk of contamination [18] . The use of three primer pairs corresponding to six target sequences in the RT-LAMP assay was conferred high specificity but can also make primer design a challenge in viruses The PRRSV RT-LAMP assay described in this study showed greater specificity than conventional RT-PCR. There was no cross-reactivity between PRRSV and other related viruses, such as CSFV strain CHLV, PCV2, PRV and SIV, which suggeated that the method was highly specific. In addition, RT-LAMP is a simple and rapid procedure, providing results within 1 h, which is significantly quicker than conventional RT-PCR. Of the 33 positive samples detected by RT-LAMP, three were missed by RT-PCR. Furthermore, restriction analysis allowed for the specific detection of highly pathogenic PRRSV strains. In summary, we have constructed a rapid, sensitive RT-LAMP assay for PRRSV detection that has the potential to be applied to on-the-spot detection in the field. Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus Development of a streptavidin-biotin immunoperoxidase procedure for the detection of porcine reproductive and respiratory syndrome virus antigen in porcine lung Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus Development of HS-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection Reproductive failure of unknown etiology Nested PCR for detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs Comparison of immunogold silver staining (IGSS) with two immunoperoxidase staining systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) antigens in formalin-fixed tissues Differentiation of North American and European porcine reproductive and respiratory syndrome virus genotypes by in situ hybridization Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus Loop-mediated isothermal amplification of DNA Passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hybridization at different times postinfection Mystery swine disease in the Netherlands: the isolation of Lelystad virus Genetic Variations of ORF5 Gene and Nsp2 Gene of Porcine Reproductive and Respiratory Syndrome Virus Isolates from China in A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus in clinical specimens