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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 establish a real-time PCR-based real-time PCR system for the detection of aureobasidium by a four-step coincidence-degenerate hybrid oligonucleotide primer (CODEHOP). Methods A pair of consensus-degenerate primers for the genus Atypical spp. Were designed. The amplification curves and melting curves of Real-time PCR were analyzed. According to the melting temperature (Tm) of primer-dimers (PDs) and amplified products, After the three-step extension step, the temperature was increased by 5 s, the temperature of the fluorescence was optimized between the PDs and the melting temperature of the specific amplification product, and the four-step CODEHOP Real-time PCR method was established. Sensitivity, specificity and repeatability. Results The Tm values of PDs ranged from 75.32 to 76.86 ℃, the Tm values of specific amplification products were 86.84 ℃ and the one-step temperature was 84 ℃. The fluorescence detection procedure can effectively remove the effect of PDs on the detection results. The specificity of this method is 100% , The detection sensitivity of virus RNA was 59.6 pg, the repeatability was good, the coefficient of variation was <5%, and the results of 12 mouse lung blind samples were in line with the expectation. Conclusion The established four-step CODEHOP real-time PCR method for detection of isodoronitis virus is sensitive and specific and can be used for the detection of murine isoviral infection.