SYBR Green I荧光RT-PCR法检测贝类中的诺如病毒

SYBR Green I real-time polymerase chain reaction for detection of Norovirus II in the shellfish

摘要:

针对诺如病毒II型的保守区域设计引物，建立了SYBR Green I实时荧光RT-PCR检测诺如病毒II型的反应体系。此方法的病毒检测下限达到102拷贝，标准曲线的线形范围为102~106拷贝，相关系数为0.9952，斜率为?2.982，截距为35.84。对诺如病毒II型检测特异，与轮状病毒、腺病毒、甲肝病毒、星状病毒无交叉反应。针对质粒标准品检测的批内试验变异系数 (CV) 为0.95％~1.69％ (n=5)，批间试验CV为0.87％~1.24％ (n=3)。运用此方法随机检测30份贝类水产品，检测出2份阳性样品。结果表明，SYBR Green I荧光RT-PCR检测诺如病毒II型的方法灵敏、特异、重复性好，可应用于贝类水产品的快速检测。

Abstract:

We set up an SYBR Green I real-time RT-PCR method for the detection of genogroup II Norovirus, and this method’s primers were encompassed the conservative region of Norovirus II. The limit of the detection was 102 copies. The standard curve’s linear range was 102–106 copies, correlation coefficient was 0.9952, the slope was ?2.982, and the intercept was 35.84. This method possessed specificity for genogroup II Norovirus, without any cross-reaction with rotavirus, adenovirus, hepatitis A virus or astrovirus. The coefficients of variation (CV) of the Ct values of the standard plasmid were 0.95%–1.69% (n=5) in intra-assay and 0.87%–1.24% (n=3) in inter-assay. We used this method to detect 30 shellfish samples, and found 3 samples were positive. This method is sensitive, specific and reliable for Norovirus II. It can be used to detect the Norovirus II in the shellfish rapidly.