CAJ | 학술논문

目的建立快速、准确、特异的定量检测百日咳杆菌的方法,并进行临床应用研究.方法根据百日咳杆菌IS481基因序列,设计并合成引物和荧光探针,建立百日咳杆菌荧光定量PCR检测方法,并对方法的特异度、重复性和灵敏度进行评价,检测百日咳疑似患者咽拭子225份和健康者咽拭子30份.结果该方法的标准曲线显示模板浓度在102～108拷贝/μl线性范围与其循环阈值(Ct)具有较好的线性关系,相关系数(r)为0.998,最小检出量为102拷贝.特异度较好,灵敏度较高,重复性实验结果显示在同一时间检测高低浓度阳性克隆质粒(2×107与3×103拷贝/μ1)10次,连续检测5次,变异系数(CV)为5.78%～16.7%.在不同时间检测高低浓度阳性克隆质粒(2×107与3×103拷贝/μl),连续7次,CV为8.25%～14.9%.经检测,疑似患者的咽拭子有41份样本为阳性,30份健康人咽拭子均为阴性.结论由于本实验建立的荧光定量PCR方法快速、灵敏度高、特异度好,适合临床实验室进行临床标本的百日咳杆菌的定量检测,有较大的应用前景.
목적 건립쾌속、준학、특이적정량검측백일해간균적방법,병진행림상응용연구.방법 근거백일해간균IS481기인서렬,설계병합성인물화형광탐침,건립백일해간균형광정량PCR검측방법,병대방법적특이도、중복성화령민도진행평개,검측백일해의사환자인식자225빈화건강자인식자30빈.결과 해방법적표준곡선현시모판농도재102～108고패/μl선성범위여기순배역치(Ct)구유교호적선성관계,상관계수(r)위0.998,최소검출량위102고패.특이도교호,령민도교고,중복성실험결과현시재동일시간검측고저농도양성극륭질립(2×107여3×103고패/μ1)10차,련속검측5차,변이계수(CV)위5.78%～16.7%.재불동시간검측고저농도양성극륭질립(2×107여3×103고패/μl),련속7차,CV위8.25%～14.9%.경검측,의사환자적인식자유41빈양본위양성,30빈건강인인식자균위음성.결론 유우본실험건립적형광정량PCR방법쾌속、령민도고、특이도호,괄합림상실험실진행림상표본적백일해간균적정량검측,유교대적응용전경.
Objective To establish a rapid,accurate,specific quantitative assay for detecting B.pertussis,and apply to clinical diagnosis.Methods According to the specific sequence of B.pertussis IS481 gene,the primers and the fluorescence probe were designed and synthesized.Then a fluorescence quantitative PCR for detecting B.pertussis was developed.The specificity,sensitivity and reproducibility of the method were evaluated.255 specimens including 225 nasopharyngeal swabs from suspected pertussis patients and 30 normal nasopharyngeal swabs were detected by fluorescence quantitative PCR.Results A rapid specific quantitative method for detecting B.pertussis was established.The standard curve of the method indicated that there was a good linear relationship between the CT value and the template concentration with the correlation coefficient being 0.998.The linear range of the system was from 102 to 108 copies/μl and the minimum was 102 copies.It had a high sensitivity and good specificity.The intra.and inter-assay coefficients of variation were 5.78%-16.7% and 8.25%-14.9% respectively.The fluorescence quantitative PCR identified 41 positive results for specimens from suspected pertussis patients and results of 30 normal specimens were all negative.Conclusions The method can quantitatively detect the B.pertussis rapidly with high sensitivity and specificity,it can be applied to clinical diagnosis.