中国人兽共患病学报2025，Vol.41Issue(12)：1240-1247,8.DOI:10.3969/j.issn.1002-2694.2025.00.182
基于媒介子探针实现对13种病原体单管检测
Single-tube detection of 13 respiratory pathogens with mediator probe technology
摘要
Abstract
This study developed a multiplex detection assay using mediator probe(MP)technology for simultaneous identifica-tion of 13 respiratory-transmitted pathogens,thus enabling rapid and accurate single-tube differential diagnosis.Thirteen respiratory pathogens were selected for detection:SARS-CoV-2,influenza A virus,influenza B virus,respiratory syncytial virus(RSV),rhino-virus,adenovirus,Mycoplasma pneumoniae,human metapneumovirus(HMPV),bocavirus,human parainfluenza virus type 3(HPIV-3),human coronavirus 229E(HCoV-229E),monkeypox virus,and enterovirus.Specific amplification primers and mediator probes were designed for each pathogen.Four universal probes labeled with Cy5 and FAM fluorescence signals were used.The Cy5 channel was used for detecting influenza B virus,influenza A virus,RSV,M.pneumoniae,adenovirus,bocavirus,and rhinovirus,whereas the FAM channel was used for detecting enterovirus,HCoV-229E,monkeypox virus,HMPV,SARS-CoV-2,and HPIV3.Pathogen identification was achieved through two-dimensional analysis of fluorescence signals and melting curves.In the Cy5 chan-nel,influenza B virus,influenza A virus,RSV,M.pneumoniae,adenovirus,bocavirus,and rhinovirus exhibited distinct amplifica-tion curves,and the melting temperatures(Tm)of the amplification products were(76.14±0.06)℃,(77.71±0.09)℃,(79.3±0.04)℃,(80.52±0.12)℃,(81.96±0.07)℃,(83.79±0.04)℃,and(84.75±0.01)℃,respectively.In the FAM channel,en-terovirus,HCoV-229E,monkeypox virus,HMPV,SARS-CoV-2,and PIV3 also demonstrated characteristic amplification curves,withTm values of(76.00±0.09)℃,(78.02±0.13)℃,(79.66±0.12)℃,(80.86±0.08)℃,(82.19±0.13)℃,and(83.44±0.16)℃,respectively.The Tm differences between amplification products within the same channel were all ≥1℃,thus enabling ef-fective discrimination.Repeatability experiments confirmed that the coefficients of variation(CV)for Tm values were consistently be-low 1％.Validation in 100 clinical samples indicated complete concordance with results obtained from a commercial fluorescence RT-PCR kit.The mediator RT-PCR method developed in this study integrates the advantages of specific and universal probes,thereby en-abling rapid and precise differential diagnosis of 13 respiratory-transmitted pathogens in a single tube through two-dimensional analy-sis of fluorescence signals and Tm.This approach may provide a robust technical foundation for early screening,prevention,and con-trol of respiratory-transmitted diseases.关键词
媒介子探针/呼吸道病原体/多重检测/熔解曲线Key words
mediator probe/respiratory pathogens/multiplex detection/melting curve分类
医药卫生引用本文复制引用
薛晓宁,张娟,徐翮飞,杜建森,张瑾..基于媒介子探针实现对13种病原体单管检测[J].中国人兽共患病学报,2025,41(12):1240-1247,8.基金项目
海关总署科技项目(No.2023HK061) Science and Technology Project of the General Administration of Customs of China(No.2023HK061) （No.2023HK061）
