Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory

BaoJ.R.; HuardT.K.; PiscitelliA.E.; TummalaP.R.; AleemiV.E.; CoonS.L.; MasterR.N.; LewinskiM.A.; ClarkR.B.

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Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory

机译：逆转录聚合酶链反应/焦磷酸测序可表征A流感2009 H1N1病毒的神经氨酸酶H275残留，以便在临床实验室中快速，特异性地检测病毒奥司他韦耐药标记

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Pandemic 2009 H1N1 is normally susceptible to oseltamivir, but variants harboring the H275Y (CAC → TAC) mutation exhibit resistance. We describe the use of a combined reverse-transcription polymerase chain reaction (RT-PCR)/pyrosequencing approach to identify the H275 residue. A total of 223 specimens were tested with this method: 216 randomly selected clinical specimens positive for 2009 H1N1 and 7 cell-culture supernatants from the Centers for Disease Control and Prevention (CDC; 4 resistant, 3 susceptible 2009 H1N1 strains). The assay detected H275Y in 1 clinical respiratory sample (0.5%) and all 4 oseltamivir-resistant strains from the CDC; the remaining 215 clinical and 3 susceptible CDC specimens were wild-type. Sanger sequencing confirmed the results for 50 of 50 selected isolates. The RT-PCR/pyrosequencing method was highly specific, producing no amplicons or valid sequences from samples containing non-H1N1 viruses or bacteria. Our findings suggest that this method provides a rapid tool for H275Y detection, with high sensitivity and potential benefit for patient care.

机译：大流行2009 H1N1病毒通常对奥司他韦敏感，但带有H275Y（CAC→TAC）突变的变体表现出抗药性。我们描述了结合使用逆转录聚合酶链反应（RT-PCR）/焦磷酸测序方法来鉴定H275残基。用此方法测试了总共223个标本：随机选择了对2009 H1N1呈阳性的临床标本和来自疾病控制与预防中心的7种细胞培养上清液（CDC; 4种抗药性，3种易感的2009 H1N1菌株）。该检测在CDC的1例临床呼吸道样本（0.5％）和所有4株耐奥司他韦的菌株中检测到H275Y。其余215个临床CDC标本和3个敏感CDC标本为野生型。 Sanger测序证实了50个分离株中有50个的结果。 RT-PCR /焦磷酸测序方法具有很高的特异性，不会从含有非H1N1病毒或细菌的样品中产生扩增子或有效序列。我们的发现表明，该方法为H275Y检测提供了一种快速工具，具有高灵敏度和对患者护理的潜在益处。

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《Diagnostic microbiology and infectious disease》 |2011年第4期|共7页
作者
BaoJ.R.; HuardT.K.; PiscitelliA.E.; TummalaP.R.; AleemiV.E.; CoonS.L.; MasterR.N.; LewinskiM.A.; ClarkR.B.;
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正文语种 eng
中图分类传染病;
关键词
2009 H1N1 virus; H275Y (H274Y) mutation; Oseltamivir resistance; Pyrosequencing;

机译：2009 H1N1病毒;H275Y（H274Y）突变;奥司他韦耐药;焦磷酸测序;

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1. Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory [J] . BaoJ.R., HuardT.K., PiscitelliA.E., Diagnostic microbiology and infectious disease . 2011,第4期

机译：逆转录聚合酶链反应/焦磷酸测序可表征A流感2009 H1N1病毒的神经氨酸酶H275残留，以便在临床实验室中快速，特异性地检测病毒奥司他韦耐药标记
2. Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase. [J] . van der Vries E, Jonges M, Herfst Journal of clinical virology: The official publication of the Pan American Society for Clinical Virology . 2010,第1期

机译：快速分子算法的评估，以检测大流行性甲型流感（H1N1）2009病毒并筛选神经氨酸酶中关键的奥司他韦耐药性（H275Y）替代。
3. A comparison of pyrosequencing and neuraminidase inhibition assays for the detection of oseltamivir-resistant pandemic influenza A(H1N1) 2009 viruses. [J] . Deng YM, Caldwell N, Hurt A, Antiviral Research . 2011,第1期

机译：焦磷酸测序和神经氨酸酶抑制试验的比较，用于检测耐奥司他韦的大流行性流感A（H1N1）2009病毒。
4. A RAPID MICRO POLYMERASE CHAIN REACTION SYSTEM (GenSpector~R Micro PCR) FOR HEP A TITIS B VIRUS DNA DETECTION [C] . Kwang W. Oh, Yoon-Kyoung Cho, Jintae Kim uTAS (Conference) . 2004

机译：一种快速的微聚合酶链式反应系统（Genpector〜R micro PCR）用于恒温B病毒DNA检测
5. Rapid detection of “Norwalk-like viruses” by reverse transcription-polymerase chain reaction (RT-PCR) and southern blot hybridisation (SBH) in outbreaks of acute gastroenteritis in eastern Ontario, Canada [D] . Wires, Shannon Meredith 2001

机译：通过逆转录聚合酶链反应（RT-PCR）和Southern blot杂交（SBH）快速检测加拿大安大略省东部急性肠胃炎暴发中的“ Norwalk样病毒”
6. Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory [O] . Jian R. Bao, Thomas K. Huard, Arelis E. Piscitelli, -1

机译：逆转录聚合酶链反应/焦磷酸测序可表征A流感2009 H1N1病毒的神经氨酸酶H275残留以便在临床实验室中快速特异性地检测病毒奥司他韦耐药标记
7. Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory [O] . Jian R. Bao, Thomas K. Huard, Arelis E. Piscitelli, 2011

机译：逆转录聚合酶链反应/焦磷酸术以表征流感A 2009 H1N1病毒的神经氨氨酸酶H275残留物，以便在临床实验室中快速和特异性检测病毒奥司他敏耐药性标志物

Reverse-transcription polymerase chain reaction/pyrosequencing to characterize neuraminidase H275 residue of influenza A 2009 H1N1 virus for rapid and specific detection of the viral oseltamivir resistance marker in a clinical laboratory

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