小西葫芦黄花叶病毒(ZYMV)的RT-PCR检测

Rapid Detection of Zucchini yellow mosaic virus by RT-PCR

小西葫芦黄花叶病毒(Zucchini yellowmosaic virus,ZYMV)是葫芦科作物上的一种重要病毒,近几年来已成了我国葫芦科作物上的一种重要病原物。根据已知的ZYMV基因组序列分别设计了2对特异性引物,在优化RT-PCR条件的基础上,成功建立了ZYMV的RT-PCR检测方法。引物ZYM1/ZYM2用于扩增整个CP基因序列(片段长度949 bp),而引物ZYM3/ZYM4用于扩增部分CP基因序列(片段长度449 bp),其中,引物ZYM3/ZYM4的检测灵敏度比引物ZYM1/ZYM2的检测灵敏度高。把所建立的方法用于南瓜(Cucurbita mos-chata)、丝瓜(Luffa cylindrica)病叶的检测,结果在这些病叶中也检出ZYMV。因此,该方法可用于ZYMV的快速、灵敏检测及分子流行病学的调查研究。

Zucchini yellow mosaic virus （ZYMV）, which is one of the most economically important viruses of cucurbit crops, has emerged as an important pathogen of cucurbits in the last few years in China. Two pairs of specific oligonucleotide primers appropriate for PCR amplicafion was designed according to the ZYMV genome sequence in GenBank. A reverse transcription- polymerase chain reaction （RT- PCR） assay was developed for the detection of ZYMV after optimization of the reaction conditions. The result showed the RT - PCR using the primer of ZYM3/ZYM4, which amplified a specific fragment about 449 bp in length including partial coat protein（CP） gene, is sensitive than using the primer of ZYM1/ZYM2, which amplified a fragment about 949 bp covering the complete CP gene. By using this method, ZYMV were detected in the diseased leaf of Luffa cylindrica and Cucurbita rnoschata. The results showed that the RT - PCR technique has provided a rapid and sensitive detection method of ZYMV, and also a technique for ZYMV molecular epidemiologie survey.