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烟草花叶病毒属病毒是作物病毒病害传入的重要途径，因此检测种子中的病毒存在对于病毒性病害的预防性防控至关重要。本研究建立了一种检测方法，可用于检测番茄（*Solanum lycopersicum*）种子中的烟草花叶病毒（Tobacco mosaic virus, TMV）、番茄花叶病毒（Tomato mosaic virus, ToMV）以及辣椒（*Capsicum annum*）种子中的辣椒轻斑驳病毒（Pepper mild mottle virus, PMMoV）。
本研究采用生物学、血清学及分子生物学手段，对不同带毒率水平的种子批开展检测分析。利用双抗体夹心酶联免疫吸附试验（DAS-ELISA）技术，可在番茄种子中检出最低带毒比例为1:170（1粒带毒种子∶总种子数）的TMV，以及最低比例为1:200的ToMV；在辣椒种子中，PMMoV的最低检测比例可达1:140。免疫捕获反转录聚合酶链式反应（IC-RT-PCR）可检出的TMV与ToMV最低比例为1:400，PMMoV的最低检测比例为1:300。将仅含1粒带毒种子的1000粒种子混合批（1:1000）拆分为30个亚批用于DAS-ELISA分析，另拆分为10个亚批用于IC-RT-PCR分析。
DAS-ELISA与IC-RT-PCR两种检测技术均可在所有分析样本中有效检出三种目标病毒，但采用反转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）及生物学试验检测烟草花叶病毒属病毒的结果可靠性欠佳。基于本研究通过拆分种子亚批以减少检测次数的方案，官方实验室常规开展的诊断检测可实现成本的大幅节约，同时保障检测的高效性与可靠性。