CAJ | 학술논문

目的建立检测非O1非O139群霍乱弧菌的多重PCR和SYBR Grcen实时PCR方法.方法分别以霍乱弧菌的外膜蛋白基因(ompW)、O1和O139群O抗原编码基因(rfb)设计引物,建立多重PCR和SYBR Green实时PCR方法,评价两种方法检测非O1非O139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O1非O139群霍乱弧菌的多重PCR和SYBR Green实时PCR方法,根据特异性条带(多重PCR)和特异性熔解温度(SYBR Green实时PCR),两种方法均能特异性检测非O1非O139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7×104 c fu/ml(多重PCR)和7×102 cfu/ml (SYBRGreen实时PCR),差异有统计学意义(P＜0.05);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0.27％～1.41％;经370株非O1非O139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O1非O139群霍乱弧菌的检测和鉴定.
목적 건립검측비O1비O139군곽란호균적다중PCR화SYBR Grcen실시PCR방법.방법 분별이곽란호균적외막단백기인(ompW)、O1화O139군O항원편마기인(rfb)설계인물,건립다중PCR화SYBR Green실시PCR방법,평개량충방법검측비O1비O139군곽란호균적특이성、중복성화일치성급최저검측균량.결과 건립료검측비O1비O139군곽란호균적다중PCR화SYBR Green실시PCR방법,근거특이성조대(다중PCR)화특이성용해온도(SYBR Green실시PCR),량충방법균능특이성검측비O1비O139군곽란호균,병능감별기타호균(5충)화장도간균(3충);량충방법적최저검측균량분별위7×104 c fu/ml(다중PCR)화7×102 cfu/ml (SYBRGreen실시PCR),차이유통계학의의(P＜0.05);대실시PCR적중복성검측,비내변이계수(CV)위0.22％～ 0.92％,비간CV위0.27％～1.41％;경370주비O1비O139군곽란호균적검측,량충방법적일치성균위100％.결론 건립적량충방법기특이성、민감성급중복성균호,가괄용우불동조건하비O1비O139군곽란호균적검측화감정.
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 rfb genes specific for both O1 and O139,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 rfb 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-O1,non-O139 V.cholerae,and to differentiate them from O1,O139 V.cholerae,other five Vibrios and 3 intestinal bacteria.The detection limits were 7 × 104 cfu/ml (multiple PCR) and 7 × 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-O1,non-O139 V.cholerae,rapidly,specifically,and reproducibly,that could all be used for the detection and identification of non-O 1,non-O 139 under different conditions.