摘要
【目的】建立一种快速、敏感、特异的检测猪细小病毒(PPV)环介导等温扩增(LAMP)方法,为诊断猪细小病毒提供准确可靠工具。【方法】根据GenBank公布的PPV序列,在其保守序列区域设计了多套LAMP引物,利用LAMP Real Time Turbidimeter LA-320仪监测反应进程并筛选最佳引物、反应条件,建立了对PPV病毒DNA进行特异扩增的LAMP检测方法,并可通过加入SYBR Green I肉眼判断结果。【结果】该方法在63℃恒温下作用45min,PPV病毒DNA获得了高效率的特异性扩增;其检出限量为0.23TCID50,敏感性高;在反应结束后加入SYBR Green I肉眼判断结果,与Real Time Turbidimeter LA-320仪监测结果一致。通过对20份临床样品的LAMP检测与免疫荧光鉴定、PCR方法比对,符合率均为20/20。【结论】本研究建立的PPV LAMP检测方法具有快速、特异、灵敏,操作简单、设备要求低的特点,适合用于临床PPV快速检测。
[Objective] The objective of the study is to establish a rapid detection of porcine parvovirus （PPV） by a loop-mediated isothermal amplification assay （LAMP）. [Method] According to the published GenBank sequences, many pairs of primers were designed targeting the conserved region of PPV. The amplification was detected with LAMP Real Time Turbidimeter LA-320. Through optimizing the LAMP primers and reaction conditions, a rapid and specific detection of PPV was established. Meanwhile, the amplified products were colored by SYBR green I after completion of the reaction, so that the amplification could be detected with naked eyes. [ Result ] The method of LAMP shows a highly efficient amplification for PPV viral target gene which performed at 63℃ for 45min by the LAMP Real Time Turbidimeter LA-320. The detection limit was 0.23 TCIDs0. The results of PPV LAMP reaction visualized the tube directly with naked eyes by the addition of SYBR Green I are consistent with the results detected by Tubidimeter real-time. Twenty clinical samples were detected by LAMP, PCR and IF, and the coincidence rate was 20/20. [ Conclusion ] PPV LAMP detection method established by the authors is rapid, specificity, high sensitivity, be easy of operation under simple conditions, which is suitable for rapid detection.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第14期3012-3018,共7页
Scientia Agricultura Sinica
基金
2010年公益性行业(农业)科研专项201003010-1
"十一五"国家科技支撑计划(2007BAD86B04-4
2006BAD06A18
2007BAD86B01)
作者简介
刘业兵，副研究员，博士。Tel：010—62103675：E-mail：liuyebing@ivdc．gov．cn
