Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA
Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA
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DOI：
10.1007/s007050170193
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发表时间：
2001-01-01
影响因子：
2.7
通讯作者：
Jorgensen, PH
中科院分区：
文献类型：
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作者：
Munch, M;Nielsen, LP;Jorgensen, PH
禽流感病毒感染是发病的主要原因，快速鉴定病毒具有重要的临床、经济和流行病学意义。我们建立了一种快速诊断禽流感病毒的单管逆转录聚合酶链反应（RT-PCR）。使用设计用于扩增NP基因的218 bp片段的引物，在单管RT-PCR中评价来自各种宿主（包括禽类、人、猪和马）的一组参考流感毒株。通过使用内部捕获探针通过PCR-ELISA检测PCR产物，确认NP甲型流感病毒来源。PCR-ELISA的敏感性比琼脂糖凝胶电泳检测PCR产物高100倍左右。RT-PCR和PCR-ELISA检测对鸡蛋中病毒传播的敏感性相当。我们还设计了用于检测流感的引物。A亚型H5和H7显示在家禽中具有致病潜力。H5引物覆盖HA基因的切割位点并特异性扩增甲型流感H5亚型。H7引物也覆盖HA切割位点，并检测到所有研究的H7参考菌株。此外，H7引物还在琼脂糖凝胶上从其他亚型扩增出非常弱和/或额外的条带。然而，H7来源和由切割位点处碱性氨基酸的存在或不存在所限定的致病潜力可以通过PCR产物的测序来确定。据我们所知，这是第一次证明RT-PCR检测的一组H7菌株只使用一个引物组。
Avian influenza virus infections are a major cause of morbidity and rapid identification of the virus has important clinical, economical and epidemiological implications. We have developed a one-tube Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for the rapid diagnosis of avian influenza A. A panel of reference influenza strains from various hosts including avian species, human, swine and horse were evaluated in a one tube RT-PCR using primers designed for the amplification of a 218 bp fragment of the NP gene. The PCR products were detected by PCR-ELISA by use of an internal catching probe confirming the NP influenza A origin. The PCR-ELISA was about 100 times more sensitive than detection of PCR products by agarose gel electrophoresis. RT-PCR and detection by PCR-ELISA is comparable in sensitivity to virus propagation in eggs. We also designed primers for the detection of the influenza. A subtypes H5 and H7 shown to have pathogenic potential in poultry. The H5 primers cover the cleavage site of the HA gene and specifically amplify influenza A subtype H5. The H7 primers also cover the HA cleavage site and detected all H7 reference strains investigated. In addition, the H7 primers also amplified very weak and/or additional bands on an agarose gel from other subtypes. However, the H7 origin and the pathogenic potential defined by the presence or absence of basic amino acids at the cleavage site can be determined by sequencing of the PCR product. As far as we know this is the first demonstration of RT-PCR detection on a panel of H7 strains using only one primer set.