建立和应用多重逆转录聚合酶链反应检测方法对全球分离的甜橙衰退病毒进行筛选。

Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

机构信息

University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, FL 33850-2299, USA.

出版信息

Phytopathology. 2010 Oct;100(10):1077-88. doi: 10.1094/PHYTO-04-10-0102.

PMID:20839943

Abstract

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

摘要

甜橙衰退病毒（CTV）基因型的不断多样化，使得检测和诊断措施变得复杂，并促使人们寻找新的分化方法。为简化 CTV 基因型的鉴定和分化，开发了一种用于 CTV 分离物筛选的多重逆转录聚合酶链反应（RT-PCR）技术。在不同 CTV 基因型的开放阅读框（ORF）-1a 内鉴定可变区，以首先开发单重（S），然后是六重（H）RT-PCR。根据核苷酸序列比较和系统发育分析，先前已将 CTV 分离物分为五个基因型（即 T3、T30、T36、VT 和 B165）。使用来自 GenBank 的核苷酸序列设计种和基因型特异性引物（GSP）。GSP 最初用于使用 S-RT-PCR 可靠地检测所有 CTV 基因型。此外，使用 H-RT-PCR 建立了对所有五个公认的 CTV 基因型的检测。根据大小鉴定了六个扩增子，一个通用所有 CTV 分离物，一个用于五个公认的基因型。基于序列分析对其进行了确认。总共对来自 29 个柑橘种植国家的 175 个 CTV 分离物进行了 S-和 H-RT-PCR 分析。其中，97 个分离物含有 T36 基因型，95 个分离物含有 T3 基因型，76 个分离物含有 T30 基因型，71 个分离物含有 VT 基因型，24 个分离物含有 B165 基因型。总共，126 个分离物含有 2 到 5 种已知 CTV 基因型的混合感染。有两个 CTV 分离物无法分配到已知基因型。与用于 CTV 基因型检测的其他方法相比，H-RT-PCR 提供了一种敏感，特异，可靠和快速的方法来筛选 CTV 基因型。有效鉴定 CTV 基因型将有助于更好地了解 CTV 分离物，包括不同基因型在引起或预防疾病中的可能相互作用。所述方法还可用于无病毒柑橘繁殖计划和 CTV 抗性品种的开发。