基于微流控芯片PCR结合免疫层析技术检测产毒型艰难梭菌方法的建立与性能评价  

Assay for detection of toxigenic Clostridioides difficile with combined microfluidic chip and immunochromatography technology

作　　者：程泓睿 宋小军 陈毓 张萌 蔡梦婷 朱坤 邰玉蕾 应士波 金大智 CHENG Hong-rui;SONG Xiao-jun;CHEN Yu;ZHANG Meng;CAI Meng-ting;ZHU Kun;TAI Yu-lei;YING Shi-bo;JIN Da-zhi(School of Laboratory Medicine,School of Bioengineering,Hangzhou Medical College,Hangzhou 310053,China;Zhejiang Provincial People’s Hospital(Affiliated People’s Hospital),Hangzhou Medical College,Laboratory Center,Hangzhou 310014,China;Zhejiang Provincial Key Laboratory of Biomarkers and In Vitro Diagnostics Translation,Hangzhou 310053,China;School of Public Health,Hangzhou Medical College,Hangzhou 310053,China;Beijing OriginGene-tech Biotechnology Co.,Ltd.,Beijing 101100,China)

机构地区：[1]杭州医学院,检验医学院、生物工程学院,杭州310053 [2]浙江省人民医院(附属人民医院),杭州医学院检验中心,杭州310014 [3]浙江省生物标志物与体外诊断转化重点实验室,杭州310053 [4]杭州医学院公共卫生学院,杭州310053 [5]北京源景泰科生物科技有限公司,北京101100

出　　处：《中国人兽共患病学报》2025年第2期142-149,共8页Chinese Journal of Zoonoses

基　　金：国家卫生计生委科学研究基金-浙江省医药卫生重大科技计划(No.WKJ-ZJ-2309);浙江省医药卫生科技计划(No.2024XY055)。

摘　　要：目的 采用微流控芯片PCR结合免疫层析技术,建立产毒型艰难梭菌检测方法,评价其性能。方法 根据艰难梭菌tcdB和tpi基因,设计引物,建立产毒型艰难梭菌微流控芯片PCR结合免疫层析检测方法,对其特异性、检测限、重复性和稳定性进行评价。收集临床腹泻患者粪便样本,平行采用Xpert C.difficile/Epi、VIDAS CDAB和本试验方法进行检测,检测结果不一致的样本进行艰难梭菌分离培养和tcdB毒素基因PCR鉴定。结果 本方法与其他腹泻相关病原菌均无交叉反应,两个浓度的tcdB和tpi基因阳性质粒(10^(5)和10^(2) copies/μL)检测重复性均为100%,且采用分别保存3、6、9和12个月的PCR试剂和制备的免疫层析试纸条进行2个浓度质粒检测,结果均呈现阳性。产毒型艰难梭菌检测限为10 copies/μL。本试验方法检测到艰难梭菌阳性33例(33/215,15.3%),与Xpert C.difficile/Epi方法检测结 的一致性(Kappa值为0.965)。以Xpert C.difficile/Epi检测结果为标准,本方法的敏感性、特异性、阳性预测值、阴性预测值分别为94.3%、100.0%、100.0%和98.9%,显著高于VIDAS CDAB方法(60.0%、98.9%、91.3%、92.7%)(Kappa=0.714,OR=157.50,95%CI:62.03~847.28,P=0.013)。结论 本试验建立的微流控芯片PCR结合免疫层析方法特异性、稳定性和重复性均良好,可快速、准确地检测临床腹泻样本中的产毒型艰难梭菌,可用于门急诊、社区医疗服务中心、流行病学现场等的艰难梭菌感染筛查。An assay was established for detection of toxigenic Clostridioides difficile by combining microfluidic chip analysis with immunochromatography,and its performance was evaluated and compared with those of the Xpert C.difficile/Epi and VIDAS CDAB tests.Primer pairs were designed according to the tcdB and tpi genes in C.difficile.The specificity,limit of detection,reproducibility,and stability were evaluated.A total of 215 stool samples from patients with diarrhea were collected and tested in parallel with the Xpert C.difficile/Epi,VIDAS CDAB,and our assay.C.difficile was isolated from samples,and the tcdB gene was identified when discrepant results were obtained from the three above assays.Our assay showed no cross-reaction with other diarrhea-associated pathogens.Its reproducibility was 100%in testing of two standard plasmids containing tcdB and tpi genes at two concentrations(10^(5) and 10^(2) copies/μL).Two standard plasmids were detected after the PCR and immunochromatography reagents had been stored for 3,6,9,and 12 months,and all the results were positive.The limit of detection was 10 copies/μL for toxigenic C.difficile.Testing of 33 samples positive for C.difficile with our assay(33/215,15.3%)yielded findings statistically coherent with those of the Xpert C.difficile/Epi test(kappa value=0.965).The sensitivity,specificity,positive predictive value,and negative predictive value of our assay,with respect to Xpert C.difficile/Epi as the standard,were 94.3%,100.0%,100.0%,and 98.9%;these values were significantly higher than those of VIDAS CDAB(60.0%,98.9%,91.3%,and 92.7%)(Kappa=0.714,OR=157.50,95%CI:62.03-847.28,P=0.013).In conclusion,our newly developed assay is specific,stable,and reproducible,and may be used for rapid and accurate detection of toxigenic C.difficile.The assay could be used for C.difficile infection screening in outpatient and emergency,community medical service center,and epidemiological settings.

关 键 词：艰难梭菌 微流控芯片 免疫层析 检测 

分 类 号：R378[医药卫生—病原生物学]