Dewey R A, Semorile L C, Grau O
Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.
J Virol Methods. 1996 Jan;56(1):19-26. doi: 10.1016/0166-0934(95)01896-4.
Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.
番茄斑萎病是一种严重病害，会影响多种具有重要经济价值的作物。从流行病学角度以及为制定成功的转基因抗性植物计划来看，必须在分子水平上区分四种已知的番茄斑萎病毒属病毒。使用与N基因互补的引物进行的逆转录聚合酶链反应（RT-PCR）分析，用于检测和区分来自不同作物及地理区域的14株阿根廷番茄斑萎病毒属分离株。提取物用耐热逆转录酶进行逆转录，PCR反应在毛细管热循环仪中进行30分钟。产物用限制性内切酶消化，鉴定出了四种所述病毒中的三种。此外，结果通过双抗夹心酶联免疫吸附测定（DAS-ELISA）得到证实。这里描述的方法快速且可靠。
