B族链球菌核酸检测试剂的性能评价

Validation of Nucleic Acid Detection Performance of Group B Streptococcus(GBS)

目的:为确保正确的选择定性分子检验的检测方法,需在方法使用前进行充分、有效的评价,以保证B族链球菌(GBS)核酸检测聚合酶链反应(PCR)-荧光探针法试剂和方法的可靠性及可行性。方法:采集临床GBS菌株20例,105 CFU/ml及10 4 CFU/ml各10例;阴道-直肠拭子样本56例,同一样本分别采用提取方法略有不同的A和B试剂盒进行PCR-荧光探针法检测。A和B试剂盒检测GBS含量为105 CFU/ml、104 CFU/ml和103 CFU/ml各1例,重复检测10次;以培养和基因测序结果为金标准,对A和B试剂盒阳性符合率、阴性符合率、总符合率、重复性、最低检测下限、可报告范围、携带污染、检测GBS特异性和抗干扰能力等进行验证和评价。结果:准确性实验中A和B试剂盒检测菌株结果与培养结果总符合率、阳性符合率和阴性符合率均为100%;56例临床拭子样本的准确性A和B试剂盒总符合率、阳性符合率分别为92.86%、76.47%和100%、100%。循环阈值(Ct)的变异系数(CV)均≤5%,B试剂盒3种含量标本的CV高于A试剂盒。A和B试剂盒最低检测下限均为103 CFU/ml。结论:B试剂盒总符合率、阳性符合率和阴性符合率均>95%,A和B试剂盒其他性能验证指标均相当,两种试剂盒均符合特异性和抗干扰实验要求。B试剂盒的GBS核酸检测试剂盒达到国内临床检测试剂性能指标要求,可以满足临床样本的检测标准。

Objective:To ensure the quantitative molecular detection method was correctly selected through got a fully and effectively evaluation before the method was applied,so as to guarantee the reliability and feasibility of reagents and methods of quantitative real-time polymerase chain reaction(RT-PCR)of Group B Streptococci(GBS)nucleic acid detection in clinical applications.Methods:20 GBS strains separated from clinical positive samples(10 5 CFU/ml 10 cases,10 4 CFU/ml 10 cases).And 56 vaginal-rectal swabs were collected.The same sample was tested by A and B kits(quantitative RT PCR)respectively.The each case of three concentrations(105CFU/ml,104CFU/ml and 103CFU/ml)of GBS was detected by A and B kit,respectively,and the number of repeated detection was 10 times for each case.And the results of bacterial culture and gene sequencing were used as the gold standard.The positive coincidence(conformity)rate,negative coincidence rate,total coincidence rate,repeatability,lowest limit of detection,reportable range,carryover,specificity and anti-interference ability of A kit and B kit in detecting GBS were verified and evaluated.Results:In accuracy tests,the total coincidence rate,positive coincidence(conformity)rate and negative coincidence rate of bacterial culture and detecting bacterial strain between A kit and B kit were 100%,respectively.And for the accuracy of 56 clinical swabs,the total coincidence rate and positive coincidence(conformity)rate of A kit and B kit were(92.86%and 72.47%:A kit)and(100%and 100%:B kit),respectively.The coefficient of variation(CV)of cycle threshold(Ct)of the two kits does not exceed 5%,while the CV of three concentrations of B kit was higher than that of A kit,respectively.The lowest limits of detection both A kit and B kit were 103CFU/ml.Conclusion:The total coincidence rate,positive coincidence rate and negative coincidence rate of B kit are more than 95%,and other performance indicators of A kit and B kit are equivalent.And both two kits meet the experimental demands of specificity and anti-interference.B kit has achieved the requirement of performance indicator of domestically clinical detection reagent,and it can meet detection standard of clinical sample.