荧光定量PCR熔解曲线法检测真菌核酸的临床应用价值评估

Clinical application evaluation of the fluorescence quantitative PCR melting curve method for detecting fungal nucleic acid

目的评估荧光定量PCR熔解曲线法检测真菌核酸的准确性及临床应用价值。方法纳入460例疑似或确诊为呼吸道真菌感染患者为研究对象,以荧光定量PCR熔解曲线法作为考核方法,以真菌26S rRNA基因核酸测定试剂盒联合Sanger测序法作为参考方法,采集各研究对象痰液样本并同步使用考核方法、参考方法检测,计算两种方法的Kappa值以评价结果的一致性。结果以参考方法为对照,考核方法检测结果总符合率为92.83%(427/460);此外,与参考方法相比,考核方法检测8种真菌(白念珠菌、光滑念珠菌、克柔念珠菌、近平滑念珠菌、热带念珠菌、新生隐球菌、季也蒙念珠菌、曲霉菌)的阳性符合率、阴性符合率、总符合率分别为97.34%(183/188)、97.06%(264/272)、97.17%(447/460);100.00%(33/33)、99.77%(426/427)、99.78%(459/460);100.00%(16/16)、99.55%(442/444)、99.57%(458/460);98.11%(52/53)、99.75%(442/444)、99.57%(458/460);95.08%(58/61)、99.50%(397/399)、98.91%(455/460);100.00%(9/9)、99.56%(449/451)、99.57%(458/460);85.00%(17/20)、99.32%(437/440)、98.70%(454/460);97.59%(81/83)、97.88%(369/377)、97.83%(450/460);两种方法检测结果一致性评价的Kappa值均大于0.8。对两种方法不一致结果进行复核检测,结果发现有53.7%(22/41)的检测结果与考核方法一致,有46.3%(19/41)的检测结果与参考方法一致。结论使用荧光定量PCR熔解曲线法能同时检测8种真菌,且检测结果与参考方法高度一致,在真菌检测中具有独特优势,且有重要的临床应用价值。

Objective To evaluate the accuracy and clinical application value of the fluorescence quantitative PCR melting curve method for detecting fungal nucleic acid.Methods 460 suspected or confirmed patients with respiratory fungal infections were enrolled in the study.The fluorescence quantitative PCR melting curve method was used as the test method,and the fungal 26S rRNA gene nucleic acid detection kit combined with Sanger sequencing was used as the reference method.Sputum samples from each study subject were collected and detected by the test method and reference method,respectively.The Kappa value of the two methods was calculated to evaluate the consistency of the results.Results Compared with the reference method,the overall conformity rate of the test method was 92.83%(427/460).Compared with the reference method,the positive conformity rates,negative conformity rates,and overall conformity rates of the test method for detecting 8 fungi,including Candida albicans,Candida glabrata,Candida krusei,Candida tropicalis,Candida parapsilosis,Cryptococcus neoformans,Candida guilliermondii,and Aspergillus,were 97.34%(183/188),97.06%(264/272),and 97.17%(447/460),100.00%(33/33),99.77%(426/427),and 99.78%(459/460),100.00%(16/16),99.55%(442/444),and 99.57%(458/460),98.11%(52/53),99.75%(442/444),and 99.57%(458/460),95.08%(58/61),99.50%(397/399),and 98.91%(455/460),100.00%(9/9),99.56%(449/451),and 99.57%(458/460),85.00%(17/20),99.32%(437/440),and 98.70%(454/460),and 97.59%(81/83),97.88%(369/377),and 97.83%(450/460),respectively.The Kappa values for the consistency evaluation of the two methods′detection results were both greater than 0.8.Upon retesting the inconsistent results of the two methods,it was found that 53.7%(22/41)of the detection results were consistent with the test method,and the others were consistent with the reference method.Conclusion The fluorescence quantitative PCR melting curve method can simultaneously detect 8 kinds of fungi,and the detection results are highly consistent with the reference method.It has unique advantages in fungal detection and important clinical application value.