基于双重芯片式数字PCR快速鉴定KPC型碳青霉烯耐药肺炎克雷伯菌的方法

Simultaneous rapid identification of blaKPC-coding carbapenemase- producing Klebsiella pneumoniae by duplex chip digital PCR

摘要:

【背景】由于碳青霉烯类药物的泛用和滥用，致使肺炎克雷伯菌碳青霉烯耐药株与日俱增，产碳青霉烯酶是肺炎克雷伯菌对碳青霉烯类药物耐药的主要原因。目前对肺炎克雷伯菌碳青霉烯耐药株的检测方法存在费时费力、特异性差、灵敏度低等问题。【目的】建立一种能同时检测肺炎克雷伯菌和碳青霉烯酶基因bla_KPC的双重芯片式数字PCR方法。【方法】依据肺炎克雷伯菌的特有基因yhaI和碳青霉烯耐药基因bla_KPC保守序列设计特异性引物和探针，确定双重芯片式数字PCR同时对yhaI和bla_KPC两个基因核酸浓度绝对定量的检测范围、检出限和最佳实验体系，并进行方法特异性、灵敏度、重复性分析及临床菌株的检测。【结果】双重芯片式数字PCR检测灵敏度比双重实时荧光定量PCR提高了约1.5个数量级，在两基因同时检出的情况下，最低检出限分别为3.74 copies/μL (yhaI基因)和1.93 copies/μL (bla_KPC基因)；优化后的双重芯片式数字PCR对参考菌株检测特异性的结果与双重实时荧光定量PCR结果一致；利用优化后的双重芯片式数字PCR方法共检测58株临床菌株，其中肺炎克雷伯菌43株，属肺炎克雷伯菌且含有bla_KPC基因的菌株13株，这与质谱及耐药谱检测结果一致。【结论】利用双重芯片式数字PCR技术建立了产KPC型碳青霉烯酶肺炎克雷伯菌的绝对定量检测方法。该方法特异性强、灵敏度高、准确度好，可用于检测具有碳青霉烯酶基因bla_KPC的肺炎克雷伯菌的核酸检测和定量分析，也为产其他类型碳青霉烯酶的病原菌检测提供了新的技术参考。

关键词:肺炎克雷伯菌;碳青霉烯酶基因bla_KPC;双重芯片式数字PCR;绝对定量;病原菌检测

Abstract:

[Background] Carbapenem-resistant Klebsiella pneumoniae strains have been on the rise with the extensive use and abuse of carbapenems. The production of carbapenemase is a common mechanism by which these strains resist killing by the carbapenems. The available detection methods for carbapenemase-producing K. pneumoniae are time- and labor-intensive, with poor specificity and sensitivity. [Objective] To develop an assay for the detection of K. pneumoniae with bla_KPC gene based on duplex chip digital PCR (duplex-cdPCR). [Methods] The specific primers and probes for the detection of the characteristic gene yhaI of K. pneumoniae and the carbapenem resistance gene bla_KPC were designed. The range of absolute quantification of nucleic acids, detection limit and optimal reaction conditions of the duplex-cdPCR for yhaI and bla_KPC were determined simultaneously. The specificity, sensitivity, and precision of the method were analyzed and the clinical strains were detected. [Results] Compared with duplex-qPCR, duplex-cdPCR had the sensitivity of about 1.5 orders of magnitude higher and the detection limit was 3.74 copies/μL (yhaI) and 1.93 copies/μL (bla_KPC), respectively. The optimized duplex-cdPCR showed the specificity consistent with that of duplex-qPCR. In this report, a total of 58 clinical strains were detected by the optimized duplex-cdPCR, of which 43 strains were detected as K. pneumoniae and 13 were K. pneumoniae harboring bla_KPC gene. This demonstrated 100% concordance with the results of mass spectrometry and drug resistance profile.[Conclusion] A duplex-cdPCR assay capable of simultaneously detecting the characteristic gene yhaI of K. pneumoniae and the carbapenem resistance gene bla_KPC in the same target was established. This assay appears to be highly specific, sensitive, and accurate. The duplex assay is suitable for nucleic acid detection and quantitative analysis of K. pneumoniae with bla_KPC gene. Moreover, it also provides a new technical reference for the detection of carbapenem-resistant pathogenic bacteria carrying other carbapenemase gene types.

Key words:Klebsiella pneumoniae;carbapenemase gene bla_KPC;duplex chip digital PCR;absolute quantification;pathogen detection