诺如病毒遗传组Ⅱ型TaqMan-MGB探针实时荧光PCR的检测研究

Detection of genotype Ⅱ of norovirus by TaqMan-MGB probe-based real-time fluorescent polymerase chain reaction

目的建立特异、灵敏的诺如病毒遗传组Ⅱ型TaqMan-MGB探针实时荧光PCR快速检测方法。方法根据GenBank诺如病毒遗传组Ⅱ型代表株保守序列设计特异引物对和TaqMan-MGB探针,调整引物、探针浓度,优化最佳反应条件,建立一步法实时荧光PCR快速检测反应体系,进行灵敏度、特异性、稳定性试验,以评价反应体系,并与引物P289/P290常规RT-PCR进行灵敏度比较。结果诺如病毒遗传组Ⅱ型TaqMan-MGB探针实时荧光PCR检测时限短,仅1h就出结果;最低检出下限为100拷贝/mL,比引物P289/P290常规RT-PCR灵敏度高100倍;与轮状病毒、腺病毒、星状病毒、甲肝病毒无交叉反应;4份核酸含量不同的标本重复检测5次,平均Ct值变异系数范围0.96%～2.27%。结论诺如病毒遗传组Ⅱ型TaqMan-MGB探针实时荧光PCR快速、特异、灵敏、稳定性好,可应用于突发公共卫生应急检测,提高快速检测能力。

Objective To establish a rapid, specific and sensitive molecular method to detect genotype Ⅱ of norovlrus using TaqMan-MGB probe-based real-time fluorescent polymerase chain reaction. Methods Specific primers and TaqMan-MGB probe were designed according to genotype Ⅱ conserved sequence of norovirus from GenBank, and then optimized concentration of primers and probe. Reaction condition was introduced to establish one step TaqMan-MGB probe-based real-time PCR system and compared the conventional RT-PCR with P 289/P 290 primers to evaluate specificity, sensitivity and stability. Results The one step TaqMan-MGB probe-based real-time PCR method found rapid and sensitive with 1 hour time limit and 100 copies/mL lowest detection limit, No cross reaction observed with other viruses including rotavirus, adenovirus, astovirus and hepatitis A virus. The coefficients of variation of the average C1 values ranged 0.96%-2.27% in 4 different virus nuclei acid concentration samples after tested repeatedly for 5 times. Conclusion The one step TaqMan-MGB probe-based real-time PCR method was rapid, specific, sensitive and stable to detect genotype Ⅱ of norovirus and had potential application in emergent response.