摘要
目的评价RNA抽提试剂的裂解缓冲液(lysis buffer,LB)对流感病毒(influenza virus,Flu-V)RNA的稳定作用。方法将流感病毒临床分离株A/Zhongshan/085/2009(H1N1)进行10-2稀释,加入RNA抽提试剂的LB后,分别置4和-20℃保存7、15、30和45 d,以未经LB处理的Flu-V稀释物作为对照。提取各样品核酸后,分别采用实时RT-PCR(靶序列长105 bp)和普通RT-PCR(扩增子长527 bp)进行检测。结果经LB处理的样品,在4和-20℃保存45 d,均能检测到特异性扩增信号;在4℃保存30 d,-20℃保存45 d,均可用于较长核酸片段的扩增分析。而未经LB处理的样品,在-20℃也可稳定45 d,用于该两种RT-PCR的检测,但在4℃仅7 d时能检测到527 bp的靶序列,30 d时,两种RT-PCR方法均未检测到靶序列。结论 LB可延缓流感病毒RNA的降解,具有用作流感病毒核酸检测样品稳定剂的潜能。
Objective To evaluate the ability of lysis buffer（LB）of RNA extraction reagent in stabilizing influenza virus（Flu-V）RNA. Methods Clinical isolate A / Zhongshan / 085 / 2009（H1N1）of Flu-V was diluted to a dilution of 10-2,then mixed well with LB of RNA extraction reagent,and stored at 4 and-20 ℃ for 7,15,30 and 45 d separately,using the diluted Flu-V untreated with LB as a control. RNA was extracted from each sample, and its quality was evaluated by both real-time RT-PCR targeting a 105 bp sequence and conventional RT-PCR targeting a 527 bp sequence. Results Specific amplification signal was detectable in the LB-treated samples after storage at 4 and-20 ℃ for 45 d. Both the samples stored at 4 ℃ for 30 d and those at-20 ℃ for 45 d were suitable for the amplification of long RNA fragments.The Flu-V suspension untreated with the LB after storage at-20 ℃ for 45 d was also stable for detection by the two RTPCR assays. However,only the target sequence at a length of 527 bp was detected in the samples after storage at 4 ℃ for7 d,while no target sequence was detected in those after storage for 30 d by two RT-PCR assays. Conclusion LB delays the degradation of Flu-V RNA,thus has a potential to be used as RNA stabilizer of influenza virus samples.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第10期1272-1274,共3页
Chinese Journal of Biologicals
基金
中山市卫生局医学科研立项课题(J2011107)
国家科技基金项目:重大专项(2012ZX10004213)资助
