植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术

Studies on the Detection of Papaya Ringspot Virus in Infected Tissue of Plant by ELISA

本研究建立和改进了检测番木瓜和西葫芦组织粗汁液里的番木瓜环斑病毒（ＰＲＶ）的ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法。用不同的ＥＬＩＳＡ方法来检测不同寄主植物粗汁液里的ＰＲＶ，其所用的合适的制备粗汁液的缓冲液是不同的。用ＤＡＣ－ＥＬＩＳＡ法检测西葫芦粗汁液时，以０．５ｍｏｌ／Ｌ磷酸盐缓冲液（ｐＨ７．５，内含０．１ｍｏｌ／Ｌ乙二胺四乙酸二钠）为宜；而检测番木瓜粗汁液时，则还要加入０．２５ｍｏｌ／Ｌ脲。用Ｄｏｔ－ＥＬＩＳＡ法检测时，在上述磷酸盐缓冲液中加入２％聚乙烯吡咯烷铜能提高对西葫芦粗汁液的检测效果。应用合适的制备粗汁液的缓冲液，ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法的灵敏度分别提高到１／４０９６和１／１０２４（稀释度）。本研究还表明，影响ＤＡＣ－ＥＬＩＳＡ法的定过测定的主要因素是粗汁液的稀释度和包被液（０．０５ｍｏｌ／Ｌ碳酸盐缓冲液，ｐＨ９．６）的用过。在较高粗汁液稀释度和包被液的用量相同时，粗汁液里的病毒含量与ＤＡＣ－ＥＬＩＳＡ法的ＯＤ４９２ｎｍ值呈真实的线性关系。

he present study established and further improved the direct antisen cuating form of indirectELISA(DAC-ELISA)and Dot-ELISA for the detection of papaya rinsspot virus(PRV)in infected tissuses of papaya(Carica papsys L.)or zucchini squash(Cucurbita pepo L.).The results showed if theELISA method to be employed or the host plant to be detected were different,then the proper phosphate buffer solution for preparing the crude plant sap to be detected should be different.With a pro-per phosphate buffer solutions for preparins the crude plaut sap to be detected,it was pirssible to increase the sensitivity of DAC-ELISA and Dot-ELISA up to a dilution of plant sap at a rate of 1/4096and 1/1024 respectively.The main factors of interfering the accuracy of quantitative measurement ofthe concentration of viral antigens in crude plant sap were the amount of carbonate coating buffer solution(0.05mol/L,pHg.6)and the dilution rate of the crude plant sap.With a hisher dilution rate ofthe plant sap and a consistent aniount of carbonate coating buffer selution,a linear relationship was established between the concentration of virus in plant sap and the optical density(OD)in the DAC-ELISA.