Nagarajan Malliga M, Simard Geneviève, Longtin Diane, Simard Carole
St-Hyacinthe Laboratory, Canadian Food Inspection Agency, 3400 Casavant Boulevard West, St-Hyacinthe, QC, Canada J2S 8E3.
J Vet Diagn Invest. 2010 May;22(3):402-8. doi: 10.1177/104063871002200309.
Because pigs are considered intermediate hosts for the emergence of novel influenza virus reassortants with associated zoonotic potential, monitoring and characterization of circulating influenza viruses in pigs are important for adequate control of infection. For this, rapid molecular diagnostic methods other than immunoassays are needed. Three novel single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) assays were developed in the current study for simultaneous detection and subtype differentiation of Influenza A virus in pigs. A conventional single-step pentaplex RT-PCR was designed for concomitant detection of the generic matrix (M) gene, hemagglutinin H1 and H3, and neuraminidase N1 and N2 genes of Swine influenza virus (SIV). In the other 2 single-step tetraplex real-time RT-PCR assays, the primers and fluorescent probes were targeted for the simultaneous detection of common M, H1, H3, and N2 SIV genes (first assay), and for M, H1, and H3 SIV genes and the H5 gene of highly pathogenic avian influenza virus of Eurasian lineage (second assay). The real-time RT-PCR assays had detection sensitivity limits ranging from 10(1) to 10(3) copies of respective in vitro RNA transcripts of M, H1, H3, H5, and N2 genes. The multiplex assays were evaluated by using SIV isolates, clinical specimens, and the appropriate synthetic template. The recent H1N1 pandemic strain isolated from pigs also was tested in simplex RT-PCR and real-time RT-PCR assays with the H1 primers and probes. The efficacy of the multiplex RT-PCR and real-time RT-PCR shows the suitability of multiplex RT-PCR and real-time RT-PCR for rapid subtype identification and monitoring in North American pigs of Influenza A virus.
由于猪被认为是具有相关人畜共患病潜力的新型流感病毒重配体出现的中间宿主，因此监测和鉴定猪体内传播的流感病毒对于充分控制感染至关重要。为此，需要免疫测定法以外的快速分子诊断方法。在本研究中开发了三种新型单步多重逆转录聚合酶链反应（RT-PCR）测定法，用于同时检测猪甲型流感病毒并进行亚型区分。设计了一种传统的单步五重RT-PCR，用于同时检测猪流感病毒（SIV）的通用基质（M）基因、血凝素H1和H3以及神经氨酸酶N1和N2基因。在另外两种单步四重实时RT-PCR测定法中，引物和荧光探针用于同时检测常见的M、H1、H3和N2 SIV基因（第一种测定法），以及M、H1和H3 SIV基因和欧亚谱系高致病性禽流感病毒的H5基因（第二种测定法）。实时RT-PCR测定法对M、H1、H3、H5和N2基因各自体外RNA转录本的检测灵敏度极限范围为10（1）至10（3）拷贝。通过使用SIV分离株、临床标本和适当的合成模板对多重测定法进行了评估。还使用H1引物和探针在单重RT-PCR和实时RT-PCR测定法中对从猪分离的近期H1N1大流行毒株进行了检测。多重RT-PCR和实时RT-PCR的有效性表明多重RT-PCR和实时RT-PCR适用于对北美猪体内的甲型流感病毒进行快速亚型鉴定和监测。
