摘要
目的建立诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR快速、特异、灵敏的检测方法,为疾病预防控制提供可靠的依据。方法根据GenBank诺如病毒遗传组Ⅰ型代表株保守序列设计特异引物对和TaqMan-MGB探针,建立一步法实时荧光RT-PCR快速检测反应体系,优化反应条件,评价反应体系的灵敏度、特异性、重复性,并与常规RT-PCR比较。结果诺如病毒遗传组Ⅰ型TaqMan-MGB探针实时荧光RT-PCR检测时限短,仅1h就出结果,与轮状病毒、腺病毒、星状病毒、甲肝病毒、诺如病毒遗传组Ⅱ型无交叉反应,最低检出下限为100拷贝/反应,比常规RT-PCR灵敏100倍,5份浓度不同的诺如病毒遗传组Ⅰ型标本重复检测5次,平均Ct值变异系数范围为0.39%～1.02%。结论诺如病毒遗传组Ⅰ型TaqMan-MGB探针实时荧光RT-PCR快速、特异、灵敏、重复性好,可应用于突发公共卫生应急检测和诺如病毒遗传组Ⅰ型监测,提高快速检测能力。
Objective To establish a rapid, specific and sensitive molecular method for the detection of genogruop I of norovirus using TaqMan-MGB probe-based real-time fluorescent polymerase chain reaction, and to provide credible molecular evidence for disease control and prevention. Method According to the genogroup Ⅰ conserved sequence of norovirus, specific primers and TaqMan-MGB probe were designed, and were optimized to establish one step real-time RT-PCR system. The specificity, sensitivity and repeatability of this system were evaluated and compared with the conventional RT-PCR. Result The one step TaqMan-MGB probe-based real-time RT-PCR for genogruop I of norovirus was so rapid that it can be reported in just an hour, and the sensitivity was 100 copies per reaction, and there was no non-specific cross reaction with other viruses, including rotavirus, adenovirus, astrovirus, hepatitis A virus and the genogruop H of norovirus. Five samples containing different concentration of norovirus were tested for 5 times, and the coefficient of variability for the average of Ct values were 0.39%-1.02%. Conclusion The rapid, specific, sensitive and repeatable one step TaqMan-MGB probe-based real-time RT-PCR for the detecting genogruop I of norovirus was successfully established, and could be applied to improve the capacity of rapid detection for the emergency process to the abrupt public hygiene affairs or regular surveillance.
出处
《热带医学杂志》
CAS
2008年第10期1030-1033,1040,共5页
Journal of Tropical Medicine
基金
广东省肇庆市科技创新计划项目(No.2006E1818)
关键词
人类杯状病毒
诺如病毒
实时荧光PCR
逆转录PCR
human calicivirus
norovirus
real-time polymerase chain reaction
reverse transcription polymerase chain reaction
