浙江农业学报
浙江農業學報
절강농업학보
ACTA AGRICULTURAE ZHEJIANGENSIS
2014年
2期
378-383
,共6页
李俊敏%周燕茹%孙宗涛%王旭%谢礼%陈剑平
李俊敏%週燕茹%孫宗濤%王旭%謝禮%陳劍平
리준민%주연여%손종도%왕욱%사례%진검평
灰飞虱%水稻条纹病毒%水稻黑条矮缩病毒%RT-PCR
灰飛虱%水稻條紋病毒%水稻黑條矮縮病毒%RT-PCR
회비슬%수도조문병독%수도흑조왜축병독%RT-PCR
Laodelphax striatellus%Rice stripe virus%Rice black-streaked dwarf virus%RT-PCR
灰飞虱传播的水稻条纹病毒（Rice stripe virus， RSV）和水稻黑条矮缩病毒（Rice black-streaked dwarf virus， RBSDV）是危害我国水稻生产最主要的2种病毒，建立灰飞虱体内RSV和RBSDV快速、可靠的检测方法是水稻生产中的重要问题。本研究根据RSV RNA3和RBSDV S10序列分别设计了2个病毒的特异性引物，通过退火温度和PCR循环数等条件的优化，建立了灰飞虱体内RSV和RBSDV快速鉴定的双重一步法RT-PCR体系。对一步法和两步法RT-PCR检测结果进行比较，表明2种方法均能准确有效鉴定灰飞虱体内的2种病毒，且两步法的检测效果略好于一步法。而灵敏度实验表明一步法RT-PCR可以从0.005 ng?μL-1的灰飞虱RNA初始模板中准确检测到病毒，完全满足单头灰飞虱体内病毒检测的需要。应用本研究建立的双重一步法RT-PCR体系对200头获毒灰飞虱样品中的RSV和RBSDV进行了检测，结果进一步证实了此方法的稳定性和可靠性。
灰飛虱傳播的水稻條紋病毒（Rice stripe virus， RSV）和水稻黑條矮縮病毒（Rice black-streaked dwarf virus， RBSDV）是危害我國水稻生產最主要的2種病毒，建立灰飛虱體內RSV和RBSDV快速、可靠的檢測方法是水稻生產中的重要問題。本研究根據RSV RNA3和RBSDV S10序列分彆設計瞭2箇病毒的特異性引物，通過退火溫度和PCR循環數等條件的優化，建立瞭灰飛虱體內RSV和RBSDV快速鑒定的雙重一步法RT-PCR體繫。對一步法和兩步法RT-PCR檢測結果進行比較，錶明2種方法均能準確有效鑒定灰飛虱體內的2種病毒，且兩步法的檢測效果略好于一步法。而靈敏度實驗錶明一步法RT-PCR可以從0.005 ng?μL-1的灰飛虱RNA初始模闆中準確檢測到病毒，完全滿足單頭灰飛虱體內病毒檢測的需要。應用本研究建立的雙重一步法RT-PCR體繫對200頭穫毒灰飛虱樣品中的RSV和RBSDV進行瞭檢測，結果進一步證實瞭此方法的穩定性和可靠性。
회비슬전파적수도조문병독（Rice stripe virus， RSV）화수도흑조왜축병독（Rice black-streaked dwarf virus， RBSDV）시위해아국수도생산최주요적2충병독，건립회비슬체내RSV화RBSDV쾌속、가고적검측방법시수도생산중적중요문제。본연구근거RSV RNA3화RBSDV S10서렬분별설계료2개병독적특이성인물，통과퇴화온도화PCR순배수등조건적우화，건립료회비슬체내RSV화RBSDV쾌속감정적쌍중일보법RT-PCR체계。대일보법화량보법RT-PCR검측결과진행비교，표명2충방법균능준학유효감정회비슬체내적2충병독，차량보법적검측효과략호우일보법。이령민도실험표명일보법RT-PCR가이종0.005 ng?μL-1적회비슬RNA초시모판중준학검측도병독，완전만족단두회비슬체내병독검측적수요。응용본연구건립적쌍중일보법RT-PCR체계대200두획독회비슬양품중적RSV화RBSDV진행료검측，결과진일보증실료차방법적은정성화가고성。
Rice stripe virus(RSV) and Rice black-streaked dwarf virus(RBSDV) are 2 important rice viruses which are transmitted by small brown planthopper (Laodelphax striatellus) and cause severe loss for rice production in China.Rapid and reliable detection of the 2 viruses in L.striatellus is a key issue for the control of these diseases . In this study, two sets of primers that are specific for RSV RNA3 and RBSDV S10 were designed, and multiple one step RT-PCR method was established for rapid detection of RSV and /or RBSDV in L.striatellus through optimized annealing temperature and cycles of PCR conditions .Comparison of one step and two step RT-PCR indicated that both methods can effectively detect RSV and/or RBSDV in L.striatellus and the result of two step RT-PCR was slightly better than that of one step .Serial dilutions assay showed that the sensitivity of one step RT-PCR method can detect the concentration as low as 0.005 ng?μL-1 , which is completely enough for the identification of viruses in single planthopper.Finally, multiple one step RT-PCR was applied to detect RSV and/or RBSDV in single insect for 200 planthoppers which further confirmed specific , sensitive and time-saving.
