摘要
目的建立检测非01非0139群霍乱弧菌的多重PCR和SYBRGreen实时PCR方法。方法分别以霍乱弧菌的外膜蛋白基因（omplV）、01和0139群O抗原编码基因（帕）设计引物，建立多重PCR和SYBRGreen实时PCR方法，评价两种方法检测非01非0139群霍乱弧菌的特异性、重复性和一致性及最低检测菌量。结果建立了检测非01非0139群霍乱弧菌的多重PCR和SYBRGreen实时PCR方法，根据特异性条带（多重PCR）和特异性熔解温度（SYBRGreen实时PCR），两种方法均能特异性检测非01非0139群霍乱弧菌，并能鉴别其他弧菌（5种）和肠道杆菌（3种）；两种方法的最低检测菌量分别为7×10。cfu／ml（多重PCR）和7×10^2cfu／ml（SYBRGreen实时PCR），差异有统计学意义（P〈O．05）；对实时PCR的重复性检测，批内变异系数（cV）为0．22％～0．92％，批间CV为O．27％～1．41％；经370株非01非0139群霍乱弧菌的检测，两种方法的一致性均为100％。结论建立的两种方法其特异性、敏感性及重复性均好，可适用于不同条件下非01非0139群霍乱弧菌的检测和鉴定。
Objective To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae （V. cholerae） serogroups non-O1 and non-O139. Methods The outer membrane protein gene （ompW） specific for V. cholerae, as well as O antigen rib genes specific for both O 1 and O 139, were used for the design of the PCR primers. Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139. Specific rib genes and ompW were developed to evaluate their specificity, limit of detection, reproducibility and consistency. Results We established multiple PCR and real-time SYBR green PCR methods. According to the specific electrophoretic bands （multiple PCR） and the specific melt curve temperature （real-time SYBR green PCR）, both methods could specifically detect the non-Ol, non-O139 V. cholerae, and to differentiate them from O 1, O 139 V. cholerae, other five Vibrios and 3 intestinal bacteria. The detection limits were 7 X 104 cfu/ml （multiple PCR） and 7 X 102 cfu/ml （real-time SYBR green PCR）, with statistically significant difference seen （P〈0.05）. For the reproducibility of real-time SYBR green PCR, the external coefficient variation ranging from 0.22% to 0.92% while the internal coefficient variation ranging from 0.27% to 1.41%. 370 strains of non-O1, non-O139 V. cholerae, were detected, with both consistency rates as 100%. Conclusion Both multiple PCR and real-time SYBR green PCR could detect non-O 1, non-O 139 V. cholerae, rapidly, specifically, and reprodueibly, that could all be used for the detection and identification of non-O 1, non-O 139 under different conditions.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2014年第1期66-70,共5页
Chinese Journal of Epidemiology
基金
基金项目：国家自然科学基金（30872260）
国家科技重大专项（2008zx10004-012）
