沙粒病毒属四步法一致-简并杂合寡核苷酸引物实时荧光PCR检测体系的建立

Development of CODEHOP 4-step program Realtime PCR to detect Arenaviruses in rodents

目的建立沙粒病毒属四步法一致-简并杂合寡核苷酸引物（CODEHOP）实时荧光PCR（Real-time PCR）检测体系。方法设计1对沙粒病毒属的一致-简并引物,分析Real-time PCR的扩增曲线和熔解曲线,根据引物二聚体（PDs）和扩增产物的熔解温度（Tm）值,在通用的三步法延伸步骤之后,增加5 s的恒温荧光检测步骤,在PDs和特异性扩增产物的熔解温度之间进行荧光检测温度的优化,建立四步法CODEHOP Real-time PCR方法,评价其灵敏度、特异性和重复性。结果 PDs的Tm值为75.32-76.86℃,特异性扩增产物的Tm值为86.84℃,增加一步温度为84℃,荧光检测步骤可有效去除PDs对检测结果的影响,该方法特异性为100%,病毒RNA检测灵敏度为59.6 pg,重复性良好,变异系数〈5%,12份鼠肺盲样的检测结果符合预期。结论建立的沙粒病毒四步法CODEHOP Real-time PCR检测方法敏感、特异,可用于鼠类沙粒病毒感染检测。

Objective To develop a consensus-degenerate hybrid oligonucleotide primer （CODEHOP） 4-step program Real-time PCR method for the detection of Arenaviruses in rodents. Methods Designed a pair of CODEHOP primers for Arenaviruses, analyzed the amplification curve and melting curve of Real-time PCR. According to the melting temperatures （Tin） of specific amplicons and primer-dimers （PDs）, CODEHOP 4-step program Real-time PCR was developed by adding a 5 s holding at the optimal temperature between Tm of PDs and amplicons for reading fluorescence signal after extension step of conventional 3-step Real-time PCR. The sensitivity, specificity and repeatability of the method were evaluated. Results Tm of PDs was between 75.32-76.86℃ and Tm of specific amplicons was 86.84℃. Adding the step which fluorescence signal reading temperature was 84℃, can effectively eliminated the artifact of PDs in CODEHOP Real-time PCR. The specificity of the method was 100% and the minimum detection to Arenaviruses RNA was 59.6 pg. The method had fine repeatability with CV value less than 5%. Twelve blind rodent lung samples were detected successfully. Conclusion The developed CODEHOP 4-step program Real- time PCR method has a high level of specificity and sensitivity, that can be effectively used for detection of different Arenaviruses carried by rodents.