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基于二维PCR技术的高毒力快速检测方法的应用与评价

Application and evaluation of a rapid detection method based on two-dimensional PCR technology for hypervirulent .

作者信息

Zhu Wenwen, Wang Yiting, Jiang Xin, Zhao Yan

机构信息

Department of Laboratory Medicine, Jinshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

出版信息

Front Microbiol. 2025 Jun 19;16:1554660. doi: 10.3389/fmicb.2025.1554660. eCollection 2025.

DOI:10.3389/fmicb.2025.1554660
PMID:40611957
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12222230/
Abstract

OBJECTIVES: Hypervirulent (hvKp) is an emerging pathogen that is more virulent than classical (cKp). This study aimed to develop an economical, high-throughput, and accurate two-dimensional polymerase chain reaction (2D-PCR) assay for the rapid detection of hvKp. MATERIALS AND METHODS: Recombinant plasmids containing the , , , and virulence genes were constructed and used for assessing the sensitivity and specificity of the 2D-PCR. Clinical samples ( = 105) were collected and evaluated the performance of the 2D-PCR to comparison with conventional PCR methods. RESULTS: The minimum detection limit of the 2D-PCR assay for , , , and were 10, 10, 10, and 10 copies/μL, respectively. Additionally, the concordance rates between the 2D-PCR and conventional PCR for detecting , , , and were all over 95%. The analysis revealed a sensitivity of 100.0% and a specificity of 96.2% when compared to conventional PCR. CONCLUSION: A 2D-PCR-based multiplex method for virulence genes of hvKp was successfully developed, demonstrating its outstanding features of high specificity, high sensitivity, and high throughput capability. This method could be used for the rapid diagnosis of infectious diseases caused by hvKp in clinical settings.

摘要

目的：高毒力肺炎克雷伯菌（hvKp）是一种新出现的病原体，其毒力高于经典肺炎克雷伯菌（cKp）。本研究旨在开发一种经济、高通量且准确的二维聚合酶链反应（2D-PCR）检测方法，用于快速检测hvKp。 材料与方法：构建了包含、、和毒力基因的重组质粒，并用于评估2D-PCR的敏感性和特异性。收集了105份临床样本，评估2D-PCR的性能，并与传统PCR方法进行比较。 结果：2D-PCR检测、、和的最低检测限分别为10、10、10和10拷贝/微升。此外，2D-PCR与传统PCR检测、、和的一致性率均超过95%。与传统PCR相比，分析显示敏感性为100.0%，特异性为96.2%。 结论：成功开发了一种基于2D-PCR的hvKp毒力基因多重检测方法，证明了其高特异性、高敏感性和高通量能力等突出特点。该方法可用于临床环境中由hvKp引起的传染病的快速诊断。

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本文引用的文献

[1]
Application of 2D polymerase chain reaction for single-tube detection of high-risk human papillomaviruses.

Front Microbiol. 2025-1-29

[2]
Sensitive and specific LAMP and multiplex qRT-PCR assays for detection of hypervirulent Klebsiella pneumoniae.

Diagn Microbiol Infect Dis. 2025-3

[3]
A rapid, sensitive, and high-throughput pathogen detection method and application of identifying pathogens based on multiplex PCR technology combined with capillary electrophoresis.

Diagn Microbiol Infect Dis. 2025-1

[4]
Clinical and laboratory insights into the threat of hypervirulent Klebsiella pneumoniae.

Int J Antimicrob Agents. 2024-9

[5]
Application of two-dimensional polymerase chain reaction to detect four types of microorganisms in feces for assisted diagnosis of IBD.

Clin Chim Acta. 2024-3-1

[6]
Impact of Multiplex PCR in the Therapeutic Management of Severe Bacterial Pneumonia.

Antibiotics (Basel). 2024-1-18

[7]
Hypervirulent Klebsiella pneumoniae: An update on epidemiology, detection and antibiotic resistance.

Acta Microbiol Immunol Hung. 2023-12-7

[8]
Identification of hypervirulent Klebsiella pneumoniae based on biomarkers and Galleria mellonella infection model.

BMC Microbiol. 2023-11-29

[9]
Hypervirulent Klebsiella pneumoniae detection methods: a minireview.

Arch Microbiol. 2023-9-6

[10]
Genetic and Functional Analysis of the Gene in Clinical Klebsiella pneumoniae Isolates.

Microbiol Spectr. 2023-8-17

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