肺炎克雷伯菌的芯片式数字PCR检测方法的建立

Development of a chip digital PCR assay for detection of Klebsiella pneumoniae

摘要:

【背景】条件致病菌肺炎克雷伯菌是医源性感染最重要的革兰氏阴性菌之一，目前对该病原菌的核酸检测方法存在费时费力、灵敏度低、准确性差等问题。【目的】建立基于芯片式数字PCR的肺炎克雷伯菌检测方法。【方法】依据肺炎克雷伯菌的16S rRNA基因保守序列设计特异性引物和TaqMan探针，通过与实时荧光定量PCR的比较分析，确定了芯片式数字PCR方法的检测范围和最佳反应条件，并进行了方法特异性、灵敏性分析及临床菌株的检测。【结果】芯片式数字PCR检测灵敏度比实时荧光定量PCR提高了约1.5个数量级，最低检出限可达到3.77 copies/μL；优化后的芯片式数字PCR特异性与实时荧光定量PCR结果一致，方法的相对标准偏差(relative standard deviation，RSD)均小于25%；本研究利用优化后的芯片式数字PCR方法共检测了28株临床菌株，检测到14株为肺炎克雷伯菌，14株为其他种属，这也与实时荧光定量PCR检测结果一致。【结论】采用芯片式数字PCR技术建立了肺炎克雷伯菌核酸检测的绝对定量方法。该方法特异性好、灵敏度高、准确度高，适合肺炎克雷伯菌的核酸检测和定量分析，也为其他临床病原菌的分子检测提供了新的技术参考。

Abstract:

[Background] Klebsiella pneumoniae is one of the major Gram-negative bacteria causing iatrogenic infection.The available methods for the nucleic acid detection of this pathogen are time-consuming and laborious and have low sensitivity and poor accuracy. [Objective] A chip digital PCR-based method for K. pneumoniae detection was established. [Methods]Specific primers and TaqMan probe were designed according to the conserved sequence of the 16S rRNA gene of K. pneumoniae.By comparison with the real-time fluorescence quantitative PCR,we determined the detection range and optimal reaction conditions of the chip digital PCR,analyzed the specificity and sensitivity of this method,and then applied this method to the detection of clinical isolates.[Results]The chip digital PCR had the limit of detection up to 3.77 copies/μL and about 1.5 orders of magnitude increase in sensitivity compared with real-time fluorescent quantitative PCR.The optimized chip digital PCR showed the specificity consistent with that of real-time fluorescence quantitative PCR,with the relative standard deviation (RSD) below 25%.Of the 28 clinical strains detected by the optimized chip digital PCR method,14 strains were identified as K. pneumoniae and 14 strains as other species,which was also consistent with the results of real-time fluorescence quantitative PCR.[Conclusion]An absolute quantitative method for nucleic acid detection of K. pneumoniae was established with the chip digital PCR.This method,characterized by good specificity,high sensitivity,and high accuracy,is suitable for nucleic acid detection and quantitative analysis of K. pneumoniae.Moreover,it provides a new technical reference for molecular detection of other clinical pathogens.