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首页> 美国卫生研究院文献>Journal of Clinical Microbiology >Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay
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Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay

机译：通过使用多重PCR和基于流体微珠的检测方法检测二十种人类呼吸道病毒的呼吸道病毒检测试剂盒的开发

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Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.
机译：病毒学实验室历来使用直接荧光抗体测定（DFA）和培养来检测六到七种呼吸道病毒。自2000年以来发现了五种新的人类呼吸道病毒之后，对检测这些新兴病毒的诊断测试的需求日益增长。我们开发了一种新的测试，可以在一次5小时的测试中检测出20种不同的呼吸道病毒类型/亚型。该检测方法采用14对病毒特异性引物对的多重PCR，然后对21种特定呼吸道病毒类型和亚型的引物进行多重靶特异性引物延伸（TSPE）反应。通过使用基于流体微球的阵列（Universal Array； TmBioscience Corporation，加拿大多伦多）和Luminex x-MAP系统对TSPE产品进行分类和鉴定。该检测法检测到甲型和乙型流感病毒；甲型流感病毒H1，H3和H5亚型（包括亚洲血统的H5N1亚型）； 1,2,3和4型副流感病毒;呼吸道合胞病毒A型和B型；腺病毒肺炎病毒鼻病毒肠病毒冠状病毒OC43、229E，严重急性呼吸系统综合症冠状病毒NL63和HKU1。在使用294项鼻咽拭子样本进行的前瞻性评估中，DFA /培养物检测到119例阳性，而呼吸道病毒小组（RVP）测试检测到112例阳性，敏感性为97％。 RVP测试还检测了另外61份阳性标本，这些标本不是DFA /培养液未检测到的，还是非DFA /培养液未检测到的病毒呈阳性的。通过使用第二种独特的PCR测定方法和组合的阳性参考标准解决了不一致的结果后，RVP测试检测到183个真实阳性中的180个，敏感性为98.5％，而DFA和培养液仅检测到183个真实阳性中的126个，灵敏度为68.8％。 RVP测试应提高医院和公共卫生实验室诊断病毒性呼吸道感染的能力，并应协助公共卫生机构确定呼吸道感染暴发的病因。

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