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采用TaqMan实时逆转录聚合酶链反应和JDV p26抗原捕获酶联免疫吸附测定法来定量体内感染急性期的杰姆布拉纳病病毒载量。

TaqMan real-time reverse transcription-PCR and JDVp26 antigen capture enzyme-linked immunosorbent assay to quantify Jembrana disease virus load during the acute phase of in vivo infection.

作者信息

Stewart Meredith, Desport Moira, Hartaningsih Nining, Wilcox Graham

机构信息

School of Veterinary and Biomedical Science, Murdoch University, South St., Murdoch, WA 6150, Australia.

出版信息

J Clin Microbiol. 2005 Nov;43(11):5574-80. doi: 10.1128/JCM.43.11.5574-5580.2005.

Abstract

Jembrana disease virus (JDV) is an acutely pathogenic lentivirus that affects Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute phase of the disease process. We report for the first time two techniques that enable quantification of the virus and the use of these techniques to quantify the virus load during the acute phase of the disease process. A one-step JDV gag [corrected] TaqMan real-time reverse transcription-PCR (RT-PCR) assay was developed for the detection and quantification of JDV RNA in plasma. The limit of detection was 9.8 x 10(2) JDV viral RNA copies over 35 cycles, equivalent to 4.2 x 10(4) JDV genome copies/ml, and a peak virus load of 1.6 x 10(12) during the acute febrile period. An antigen capture enzyme-linked immunosorbent assay (ELISA) was also developed to quantify the levels of JDV capsid (JDVp26) over a linear range of 10 to 200 ng/ml. Viral RNA and JDVp26 levels were correlated in 48 plasma samples obtained from experimentally infected cattle. A significant positive correlation (R = 0.860 and r(2) = 0.740) was observed between the two techniques within the range of their detection limits. The relatively insensitive capture ELISA provides an economical and feasible method for monitoring of virus in the absence of more sensitive techniques.

摘要

杰姆巴纳病病毒(JDV)是一种急性致病性慢病毒,可感染印度尼西亚的巴厘牛。由于无法在体外繁殖该病毒,因此难以对JDV进行定量,并确定疾病急性期病毒复制的动力学。我们首次报告了两种能够对病毒进行定量的技术,以及在疾病急性期使用这些技术对病毒载量进行定量的情况。开发了一种一步法JDV gag [校正后] TaqMan实时逆转录聚合酶链反应(RT-PCR)检测方法,用于检测和定量血浆中的JDV RNA。检测限为35个循环内9.8×10²个JDV病毒RNA拷贝,相当于4.2×10⁴个JDV基因组拷贝/毫升,在急性发热期病毒载量峰值为1.6×10¹²。还开发了一种抗原捕获酶联免疫吸附测定(ELISA),用于在10至200 ng/ml的线性范围内定量JDV衣壳(JDVp26)水平。从实验感染的牛身上采集的48份血浆样本中,病毒RNA水平和JDVp26水平具有相关性。在两种技术的检测限范围内,观察到显著的正相关(R = 0.860,r² = 0.740)。在缺乏更灵敏技术的情况下,相对不灵敏的捕获ELISA为监测病毒提供了一种经济可行的方法。

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