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
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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DOI：
10.1128/jcm.02436-06
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发表时间：
2007-09-01
影响因子：
9.4
通讯作者：
Janeczko, R.
中科院分区：
文献类型：
--
作者：
Mahony, J.;Chong, S.;Janeczko, R.
病毒学实验室历史上使用直接荧光抗体测定（DFA）和培养来检测六种或七种呼吸道病毒。自2000年以来，发现了五种新的人类呼吸道病毒，因此越来越需要诊断测试来检测这些新出现的病毒。我们开发了一种新的检测方法，可以在一次5小时的检测中检测出20种不同的呼吸道病毒类型/亚型。该检测试剂盒使用14种病毒特异性引物对进行多重PCR，然后使用21种针对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，多伦多，加拿大）和Luminex x-MAIP系统分选和鉴定TSPE产物。该检测试剂盒可检出甲型和B型流感病毒;甲型流感病毒亚型111、113和H5（包括亚洲谱系的亚型115 N1）;副流感病毒1、2、3和4型;呼吸道合胞病毒A和13型;腺病毒;偏肺病毒;鼻病毒;肠病毒;和冠状病毒OC 43、229 E、严重急性呼吸综合征冠状病毒、NL 63和HKUL在使用294个鼻咽拭子样本的前瞻性评价中，DFA/培养检出119例阳性，呼吸道病毒盘（RVP）检测检出1112例阳性，灵敏度为97%。RVP检测还检测到另外61份阳性标本，这些标本要么未被DFA/培养检出，要么对DFA/培养未检测的病毒呈阳性。在通过使用第二种独特的PCR检测和使用阳性的组合参考标准解决不一致结果后，RVP检测检测到183个真阳性中的180个，灵敏度为98.5%，而DFA和培养仅检测到183个真阳性中的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原体。
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-MAIP system. The assay detected influenza A and B viruses; influenza A virus subtypes 111, 113, and H5 (including subtype 115N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and 13; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKUL In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 1112 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.