Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene.
Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene.
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DOI：
10.1186/s12879-023-08240-w
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发表时间：
2023-05-25
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
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随着输血安全问题日益成为公众关注的焦点，建立一种快速、敏感、特异、经济的多重PCR方法，用于同时检测B肝炎病毒（HBV）、丙型肝炎病毒（HCV）、戊型肝炎病毒（HEV）和梅毒螺旋体（T.在血液中的pallidum）是至关重要的。针对目的基因保守区设计了5对引物和探针，建立了一步法五重实时荧光定量PCR（qRT-PCR）检测HBV、HCV、HEV、T.苍白球和RNase P（管家基因），提供样本质量检查。对浙江省2400份献血员和患者血样进行检测，并与市售单重qPCR和血清学检测方法进行比较。HBV、HCV、HEV和T.苍白球的拷贝数分别为7.11拷贝/微升、7.65拷贝/微升、8.45拷贝/微升和9.06拷贝/微升。该方法具有良好的特异性和精密度。与单重qPCR检测相比，检测HBV、HCV、HEV和T.苍白球表现出100%的临床敏感性，特异性和一致性。2400份血样中HBsAg阳性2份（0.08%），抗-HCV阳性3份（0.13%），抗-HEV IgM阳性29份（1.21%），抗-T阳性6份（0.25%）。梅毒阳性标本核酸检测证实阴性。血清学检测HBVDNA阳性和HEVRNA阳性各1例（分别为0.04%和0.04%）。开发的五重qRT-PCR是第一种同时、灵敏、特异和可重复检测HBV、HCV、HEV、T的检测方法。苍白球和核糖核酸酶P。该方法可在感染窗口期检测血液中的病原体，为有效筛查献血者和临床早期诊断提供了良好的工具。
With the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial. Five primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays. The 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing. The developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.
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