Procedure for detecting tobamovirus in tomato and pepper seeds decreases the cost analysis

ABSTRACT The transmission of tobamovirus by tomato and pepper seeds is an important mean of virus introduction in crops. Therefore, detecting its presence in the seed becomes essential for the preventive control of virus diseases. In this study, a method was proposed for the detection of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in tomatoes (Solanum lycopersicum) and Pepper mild mottle virus (PMMoV) in pepper (Capsicum annum) seeds. Seed lots with different levels of incidence were analyzed by biological, serological, and molecular methods. Using DAS-ELISA technique, it was possible to detect TMV up to the limit rate of 1:170 (1 contaminated seed: total seeds) and the ToMV up to 1:200 in tomato seeds; PMMoV in pepper seeds was detected up to 1:140. The IC-RT-PCR detected the TMV and ToMV up to the limit of 1:400 and PMMoV up to the limit of 1:300. The assembled lots containing only 1 contaminated seed in 1000 (1:1000) were combined into 30 sub lots for DAS-ELISA analysis and 10 sub lots for IC-RT-PCR analysis. Both techniques, DAS-ELISA and IC-RT-PCR, were efficient to detect the three viruses in all analyzed samples, but the detection of tobamoviruses with RT-PCR and biological tests was not reliable. Based on the results of this study, in which a combination of seeds in sub lots was made to reduce the number of tests performed, it is possible to make significant savings in the cost of the diagnostic methods routinely conducted in official laboratories, with high efficiency and reliability.摘要 番茄与辣椒种子携带传播烟草花叶病毒属（tobamovirus）病毒，是作物病毒病传入的重要途径。因此，检测种子中该类病毒的存在，对病毒病的预防性防控至关重要。本研究建立了一种检测番茄（Solanum lycopersicum）种子中烟草花叶病毒（Tobacco mosaic virus, TMV）、番茄花叶病毒（Tomato mosaic virus, ToMV），以及辣椒（Capsicum annum）种子中辣椒轻斑驳病毒（Pepper mild mottle virus, PMMoV）的方法。本研究采用生物学、血清学及分子生物学方法，对不同带毒率的种子批进行了检测分析。采用双抗体夹心酶联免疫吸附测定（DAS-ELISA）技术，可在番茄种子中检测到最低带毒比例为1:170（1粒染毒种子:总种子数）的TMV，以及1:200的ToMV；在辣椒种子中可检测到最低带毒比例为1:140的PMMoV。免疫捕获反转录聚合酶链式反应（IC-RT-PCR）对TMV和ToMV的最低检测限可达1:400，对PMMoV的最低检测限可达1:300。将仅含1粒染毒种子的1000粒种子混合批次（1:1000）进一步划分为30个亚批次用于DAS-ELISA检测，以及10个亚批次用于IC-RT-PCR检测。DAS-ELISA与IC-RT-PCR两种技术均可高效检测所有受试样本中的三种病毒，但采用反转录聚合酶链式反应（RT-PCR）及生物学试验检测烟草花叶病毒属病毒的结果并不可靠。基于本研究通过将种子混合为亚批次以减少检测次数的结果，官方实验室可在日常诊断检测中大幅降低成本，同时保持高效性与可靠性。