Sains Malaysiana 54(3)(2025): 757-767

http://doi.org/10.17576/jsm-2025-5403-12

A TaqMan Duplex Quantitative PCR Method for Detecting Klebsiella pneumoniae has been Developed Based on Pan-Genome Analysis

(Kaedah PCR Kuantitatif Dupleks TaqMan untuk Mengesan Klebsiella pneumoniae telah Dibangunkan Berdasarkan Analisis Pan-Genom)

ZENGHUI LI, MINGMING HUA, XINYU LIU, YI WANG, MENGMENG KONG, ZHENG HU & BO YANG*

A National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), School of Life and Health Sciences, Hubei University of Technology, Wuhan 430070, China

Received: 2 September 2024/Accepted: 26 November 2024

Abstract

Klebsiella pneumoniae is a significant pathogen capable of causing infections in the respiratory, urinary, and bloodstream. In this study, bioinformatics-based pan-genome analysis identified specific and conserved LptD and MerR protein gene sequences in K. pneumoniae. Based on these sequences, specific primers and probes were designed, and the reaction system and conditions were optimized to establish a TaqMan probe-based duplex real-time quantitative PCR method for detecting K. pneumoniae. The method was tested on genomic DNA from 14 common pathogens and negative controls. The results showed that only the genomic DNA of K. pneumoniae was positive, while all other samples were negative. The detection limits for LptD and MerR gene-positive standard plasmid DNA were 5.28 × 10¹ copies/μL and 5.78 × 10¹ copies/μL, respectively, and the coefficient of variation for Ct values between intra- and inter-gene groups was less than 3%. These results indicate that the established TaqMan probe-based duplex real-time quantitative PCR method can specifically and rapidly detect K. pneumoniae, which is of significant importance for clinical diagnosis and treatment.

Keywords: Duplex quantitative PCR; Klebsiella pneumoniae; LptD gene; MerR gene; pan-genome analysis

Abstrak

Klebsiella pneumoniae adalah patogen yang signifikan dan mampu menyebabkan jangkitan pada sistem pernafasan, sistem kencing dan aliran darah. Dalam kajian ini, analisis pan-genom berasaskan bioinformatik telah mengenal pasti urutan gen protein MerR dan LptD yang khusus dan terpelihara dalam K. pneumoniae. Berdasarkan urutan ini, primer dan prob khusus telah direka bentuk dan sistem serta syarat reaksi telah dioptimumkan untuk membangunkan kaedah PCR kuantitatif masa nyata dwi-fluoresen TaqMan untuk mengesan K. pneumoniae. Kaedah ini telah diuji pada DNA genom daripada 14 patogen biasa dan kawalan negatif. Keputusan menunjukkan bahawa hanya DNA genom K. pneumoniae yang didapati positif, manakala semua sampel lain adalah negatif. Had pengesanan untuk DNA plasmid piawai positif gen LptD dan MerR masing-masing adalah 5.28 × 10¹ salinan/μL dan 5.78 × 10¹ salinan/μL dan pekali variasi untuk nilai Ct antara kumpulan intra- dan inter-gen adalah kurang daripada 3%. Keputusan ini menunjukkan bahawa kaedah PCR kuantitatif masa nyata dwi-fluoresen TaqMan yang dibangunkan boleh mengesan K. pneumoniae secara khusus dan pantas, yang mempunyai kepentingan besar untuk diagnosis dan rawatan klinikal.

Kata kunci: Analisis pan-genom; gen MerR; gen LptD; Klebsiella pneumoniae; PCR kuantitatif dwi

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*Corresponding author; email: yangbo@hbut.edu.cn