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

IF 2.1 4区医学 Q3 INFECTIOUS DISEASES

Diagnostic microbiology and infectious disease Pub Date : 2024-10-20 DOI:10.1016/j.diagmicrobio.2024.116563

ZhiYong Pan , Wei Wang , Chen Zhang , LiangWei Mao , YiRong Li , LinLing Yuan , ZhiQiang Li

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Abstract

Respiratory infections are one of the leading causes of death worldwide. Rapid and accurate detection of pathogens is crucial for timely treatment. This study established a detection method based on multiplex PCR combined with capillary electrophoresis, capable of simultaneously detecting 28 common respiratory pathogens. We used specially designed homo-tag primers, significantly reducing the formation of primer dimers and improving the specificity and sensitivity of amplification. After two rounds of PCR amplification, the products were subjected to fragment analysis using the ABI 3500XL Genetic Analyzer, enabling automated result interpretation. The detection limit of this method reaches 2.77 × 10² copies/ml, with some pathogens reaching as low as 2.77 × 10 copies/ml. The detection of 147 clinical specimens showed that the overall consistency of this method with culture, colloidal gold, fluorescent quantitative PCR, and NGS was 75.5 %. The multiplex PCR detection method established in this study is rapid, sensitive, and high-throughput, can be used for routine screening of common pathogens in respiratory infections, providing an important basis for clinical diagnosis and treatment.

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基于多重 PCR 技术与毛细管电泳相结合的快速、灵敏、高通量病原体检测方法及其在识别病原体方面的应用

呼吸道感染是导致全球死亡的主要原因之一。快速准确地检测病原体对及时治疗至关重要。本研究建立了一种基于多重 PCR 结合毛细管电泳的检测方法，能够同时检测 28 种常见的呼吸道病原体。我们使用了专门设计的同标签引物，大大减少了引物二聚体的形成，提高了扩增的特异性和灵敏度。经过两轮 PCR 扩增后，使用 ABI 3500XL 基因分析仪对扩增产物进行片段分析，从而实现结果的自动解读。该方法的检测限达到 2.77 × 102 拷贝/毫升，部分病原体的检测限低至 2.77 × 10 拷贝/毫升。对 147 份临床标本的检测结果表明，该方法与培养、胶体金、荧光定量 PCR 和 NGS 的总体一致性为 75.5%。该研究建立的多重 PCR 检测方法快速、灵敏、高通量，可用于呼吸道感染常见病原体的常规筛查，为临床诊断和治疗提供重要依据。

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来源期刊

Diagnostic microbiology and infectious disease 医学-传染病学

CiteScore

5.30

自引率

3.40%

发文量

149

审稿时长

56 days

期刊介绍： Diagnostic Microbiology and Infectious Disease keeps you informed of the latest developments in clinical microbiology and the diagnosis and treatment of infectious diseases. Packed with rigorously peer-reviewed articles and studies in bacteriology, immunology, immunoserology, infectious diseases, mycology, parasitology, and virology, the journal examines new procedures, unusual cases, controversial issues, and important new literature. Diagnostic Microbiology and Infectious Disease distinguished independent editorial board, consisting of experts from many medical specialties, ensures you extensive and authoritative coverage.