Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR
Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR
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DOI：
10.1128/jcm.38.3.1191-1195.2000
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发表时间：
2000-03-01
影响因子：
9.4
通讯作者：
Echevarria, JE
中科院分区：
文献类型：
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作者：
Aguilar, JC;Pérez-Breña, MP;Echevarria, JE
我们描述了一种能够检测和区分所有已知的人类副流感病毒(HPIV)的多重逆转录-聚合酶链式反应(m-RT-PCR)方法。与细胞培养分离法和m-RT-PCR法比较的参考菌株的一系列稀释实验显示，对HPIV4B型(HpN-4B)的敏感性从0.0004%组织培养感染剂量(TCID_(50))到对HPIV-3的32TCID(50)S。在所有病例中，最少可检测到10个含有HPIV扩增产物的质粒。对201例下呼吸道疾病住院患儿的鼻咽标本进行m-RT-PCR检测，发现分离的HPIV有24株HPIV-3、23株HPIV-1、10株HPIV-4和7株HPIV-2，而只有42株HPIV-1、14株HPIV-3、6株HPIV-2和1株HPIV-4在细胞培养中生长。我们的m-RT-PCR检测HPIV感染比细胞培养分离或间接免疫荧光检测HPIV感染更敏感。此外，在本研究中，HPIV-4比HPIV-2更频繁地被检测到，这表明由于细胞培养的不敏感性，它作为一种下呼吸道病原体可能被低估了。
We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID50) for HPIV type 4B (HPN-4B) to 32 TCID(50)s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.