利用TAS-ELISA和免疫捕获RT-PCR检测黄瓜花叶病毒分离物并进行亚组分析
Detection and subgrouping of Cucumber mosaic virus isolates by TAS-ELISA and immunocapture RT-PCR.
作者信息
Yu Cui, Wu Jianxiang, Zhou Xueping
机构信息
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, PR China.
出版信息
J Virol Methods. 2005 Feb;123(2):155-61. doi: 10.1016/j.jviromet.2004.09.014.
Eight mouse hybridoma cell lines secreting monoclonal antibodies (MAbs) against Cucumber mosaic virus (CMV) were produced. Analysis of the specificities of the MAbs against CMV isolates by triple antibody sandwich (TAS)-ELISA demonstrated that four MAbs were specific for subgroup I (S-I) isolates and two for subgroup II (S-II) isolates, whereas another two MAbs could detect both S-I and S-II isolates. TAS-ELISA and immunocapture RT-PCR (IC-RT-PCR) methods were then established for reliable and efficient detection and subgrouping of CMV isolates using the produced MAbs. When 197 field samples collected from six provinces in China were tested by TAS-ELISA, 130 samples were found to be infected by CMV. Among them, 121 samples were infected by S-I isolates (93.1%) and another nine samples by S-II isolates (6.9%). In IC-RT-PCR using the MAbs and specific primers in the region of the coat protein (CP) gene, samples shown to contain S-I isolates by TAS-ELISA gave one specific band about 500 nucleotides in length, whereas samples containing S-II isolates produced a single band with the length of approximately 600 nucleotides. The validity and reliability of the results of TAS-ELISA and IC-RT-PCR was confirmed by sequencing and phylogenetic analysis of nearly full-length CP genes of the isolates.
制备了8株分泌抗黄瓜花叶病毒（CMV）单克隆抗体（MAb）的小鼠杂交瘤细胞系。通过三抗体夹心（TAS）-ELISA分析这些单克隆抗体对CMV分离株的特异性，结果表明，4株单克隆抗体对I亚组（S-I）分离株具有特异性，2株对II亚组（S-II）分离株具有特异性，而另外2株单克隆抗体可同时检测S-I和S-II分离株。随后建立了TAS-ELISA和免疫捕获RT-PCR（IC-RT-PCR）方法，以利用所产生的单克隆抗体对CMV分离株进行可靠、高效的检测和亚组分类。当用TAS-ELISA检测从中国6个省份采集的197份田间样本时，发现130份样本感染了CMV。其中，121份样本感染了S-I分离株（93.1%），另外9份样本感染了S-II分离株（6.9%）。在使用单克隆抗体和外壳蛋白（CP）基因区域的特异性引物进行的IC-RT-PCR中，经TAS-ELISA显示含有S-I分离株的样本产生了一条长度约为500个核苷酸的特异性条带，而含有S-II分离株的样本产生了一条长度约为600个核苷酸的单一条带。通过对分离株CP基因近全长序列的测序和系统发育分析，证实了TAS-ELISA和IC-RT-PCR结果的有效性和可靠性。
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