苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒，能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清，建立了高效、准确的AMV间接ELISA检测方法，并应用于病害调查和抗性鉴定，结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍，血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片，不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测，有46份样品检测结果一致，符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明，病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定，结果显示40份大豆全部感染AMV,但是病毒载量存在差异，部分品种表现出AM...

Establishment of ELISA for alfalfa mosaic virus and its application in soybean virus investigation and resistance identification of germplasm resources

Alfalfa mosaic virus (AMV) is a plant virus with worldwide distribution, wide host range, and serious harmfulness, which causes serious disease in soybean. In this study, the antiserum prepared by prokaryotic expression of AMV CP protein was used to establish an efficient and accurate indirect ELISA method for detection of AMV, which was applied to disease investigation and resistance identification. The results showed that the titers of the prepared three antiserums against recombinant protein and crude extract of soybean plants infected with AMV reached 256 000 times. Serum specificity analysis showed that the three antiserums only recognized AMV infected soybean leaves and did not recognize soybean mosaic virus (SMV) infected soybean leaves. A total of 50 soybean samples suspected to be infected with AMV were simultaneously detected by the established AMV indirect ELISA and conventional RT-PCR methods. The detection results of 46 samples were consistent, and the coincidence rate was 92%. The established AMV ELISA method and SMV ELISA method were used to detect the virus in soybean samples from the main soybean producing areas in Jilin province. The results showed that the detection rate of virus was 38.30%, the positive rate of SMV was 30.85%, the positive rate of AMV was 17.06%, and the composite infection rate was 9.61%. Resistance identification of 40 soybean varieties inoculated with AMV showed that all 40 soybean varieties were infected with AMV, but the viral load was significantly different. Some varieties showed AMV resistance, including 11 soybean resistant resources. The soybean disease caused by AMV had become one of the main diseases of soybean in Jilin province for the first time.