南方水稻黑条矮缩病毒一步双重RT-PCR 检测技术及其应用

 An one-step dual RT-PCR method was developed for Southern rice black-streaked dwarf virus (SRBSDV) detection from host plants and insect vector white-backed planthopper (Sogatella furcifera Horvath,Hemiptera: Delphacidae), from which two cDNA fragments of the viral genome S5 and S10 were amplified
simultaneously. Two primer pairs, S5-F1 / S5-R2 (5′-ttacaactggagaagcattaacacg-3′ / 5′-atgaggtattgcgtaactgagcc -3′) and S10-oF / S10-oR(5cgcgtcatctcaaactacag -3′ / 5′-tttgtcagcatctaaagcgc -3′), were selectedfrom 40 primer pairs based on SRBSDV genome sequences, and amplified viral S5 ORF1 fragment (819 bp) and cp gene fragment ( 682 bp), respectively. Using total RNA extracts from infected plant leaf tissue or individual planthopper adult as templates, one step RT-PCR reaction in the conditions of annealing temperature of 53℃ with the final concentrations of 240 nmol / L S5-F1 / S5-R2 and 120 nmol / L S10-oF / S10-oR generated a similar yield of two amplicons in argrose electrophoresis analysis. Sequence analysis confirmed the correct
result of the amplification. Additionally, several commercal RNA extraction kits was proven to be fit for the template preparation, and the protocol for total RNA extraction from plant leaf tissue and individual planthopper was simplified by sample grinding direct in eppendorf tube. This method can be used for SRBSDV detection of dried or 70% alcochol soaked planthopper samples stored over one month in room temprature. Key words: Southern rice black-streaked dwarf virus; Sogatella furcifera Horvath

Detection of Southern rice black-streaked dwarf virus using one-step dual RT-PCR

An one-step dual RT-PCR method was developed for Southern rice black-streaked dwarf virus(SRBSDV) detection from host plants and insect vector white-backed planthopper(Sogatella furcifera Horvath,Hemiptera: Delphacidae),from which two cDNA fragments of the viral genome S5 and S10 were amplified simultaneously.Two primer pairs,S5-F1/S5-R2(5′-ttacaactggagaagcattaacacg-3′/5′-atgaggtattgcgtaactgagcc-3′) and S10-oF/S10-oR(5′-cgcgtcatctcaaactacag-3′/5′-tttgtcagcatctaaagcgc-3′),were selected from 40 primer pairs based on SRBSDV genome sequences,and amplified viral S5 ORF1 fragment(819 bp) and cp gene fragment(682 bp),respectively.Using total RNA extracts from infected plant leaf tissue or individual planthopper adult as templates,one step RT-PCR reaction in the conditions of annealing temperature of 53℃ with the final concentrations of 240 nmol/L S5-F1/S5-R2 and 120 nmol/L S10-oF/S10-oR generated a similar yield of two amplicons in argrose electrophoresis analysis.Sequence analysis confirmed the correct result of the amplification.Additionally,several commercal RNA extraction kits was proven to be fit for the template preparation,and the protocol for total RNA extraction from plant leaf tissue and individual planthopper was simplified by sample grinding direct in eppendorf tube.This method can be used for SRBSDV detection of dried or 70% alcochol soaked planthopper samples stored over one month in room temprature.