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

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.

Key words:Klebsiella pneumoniae;chip digital PCR;absolute quantification;pathogen detection