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首页> 美国卫生研究院文献>Elsevier Public Health Emergency Collection >Development of a real-time RT-PCR assay for detection of resistance to oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleotide polymorphism probes
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Development of a real-time RT-PCR assay for detection of resistance to oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleotide polymorphism probes

机译：使用单核苷酸多态性探针开发实时RT-PCR测定法以检测2009年甲型流感大流行（H1N1）病毒对奥司他韦的耐药性

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Resistance to oseltamivir in pandemic (H1N1) 2009 influenza A virus is linked to an amino acid change from histidine (H) to tyrosine (Y) at position 275 in the neuraminidase protein (NA). A real-time one step RT-PCR assay using single nucleotide polymorphism (SNP) probes was developed to detect this mutation in respiratory specimens. The limit of detection was 47.6 copies/reaction for wild-type H275 RNA and 52.9 copies/reaction for the mutant H275Y RNA. The assay did not cross-react with other respiratory pathogens. The clinical sensitivity and specificity of the assay was compared to the gold standard Sanger sequencing method using 25 sensitive, 15 resistant and 20 negative samples. The sensitivity and specificity was 88.0% and 100% respectively with the SOIV_Osel_SEN probe designed to detect the H275 allele and 100% for the SOIV_Osel_RES probe detecting the 275Y allele. The sensitivity of the assay using nine admixtures of sensitive and resistant alleles was 88.9% and 77.8% with the SOIV_Osel_SEN probe and SOIV_Osel_RES probe respectively. The presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro RNA were detected reproducibly. This assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing.
机译：在2009年甲型H1N1大流行性流感病毒中对奥司他韦的抗性与神经氨酸酶蛋白（NA）中第275位的氨基酸从组氨酸（H）变为酪氨酸（Y）的变化有关。开发了使用单核苷酸多态性（SNP）探针的实时一步RT-PCR测定法，以检测呼吸道标本中的这种突变。野生型H275 RNA的检出限为47.6拷贝/反应，突变型H275Y RNA的检出限为52.9拷贝/反应。该测定未与其他呼吸道病原体发生交叉反应。使用25个敏感，15个耐药性和20个阴性样品，将该试验的临床敏感性和特异性与金标准Sanger测序方法进行了比较。设计用于检测H275等位基因的SOIV_Osel_SEN探针的灵敏度和特异性分别为88.0％和100％，用于检测275Y等位基因的SOIV_Osel_RES探针的灵敏度和特异性分别为100％。使用SOIV_Osel_SEN探针和SOIV_Osel_RES探针的9种敏感和抗性等位基因混合物的测定的灵敏度分别为88.9％和77.8％。可重复检测患者样品中混合的敏感和耐药等位基因以及体外RNA混合物的存在。该测定法可用于筛选对奥司他韦具有抗药性的原始样品，而无需进行培养和表型测试。

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