复合探针荧光定量PCR法检测鼠疫耶尔森氏菌的实验研究

    李禾，吴忠华，郑伟，吕沁风

    浙江国际旅行卫生保健中心，浙江 杭州 310003

    摘要：目的 建立用复合探针荧光定量PCR快速检测鼠疫耶尔森氏菌的方法。方法研究根据鼠疫耶尔森氏菌pla基因的核苷酸序列设计特异引物，通过PCR法的特异性、灵敏度和重复性研究，建立了复合探针荧光定量PCR检测鼠疫耶尔森氏菌的方法，用于鼠疫的筛选和诊断。结果该检测方法的特异性为100%，最低可检出10个拷贝的质粒DNA分子，可对1×101~1×106拷贝范围内的模板进行定量，最低可检测至1×102 cfu/ml细菌。该方法的精密度好，阳性质控品和阴性质控品不同时间测定3次及同一时间5次重复实验结果Cv值均＜5%。结论本研究建立的复合探针实时荧光定量PCR检测鼠疫耶尔森氏菌的方法，可对鼠疫耶尔森氏菌进行快速检测、准确辨别和早期诊断，对鼠疫的早期快速诊断具有重要意义。

    关键词：复合探针;荧光定量PCR;鼠疫耶尔森氏菌

    中图分类号：R516.8　文献标识码：B

    Research on Yersinia pestis detection byfluorescent quantitative

    PCR with composite probe

    LI He, WU Zhong-hua, ZHENG Wei, LVQin-feng

    Zhejiang International Travel HealthcareCenter, Hangzhou, Zhejiang 310003,China

    Abstract:   Objective  To establish fluorescent quantitative PCR assay with compositeprobe for Yersinia pestis detection.  Methods   Pla gene nucleotide sequences of Yersiniapestis was used for the design of PCR primers. According to studythe specificity, sensitivity and reproducibility of primers by PCRmethod, a composite probe fluorescent quantitative PCR assay forYersinia pestis detection was established for screening anddiagnosis of plague.  Results   The specificity ofcomposite probe fluorescent quantitative PCR assay was 100%. Theassay can detect the minimum 10 copies of plasmid DNA molecules.Accurate quantization can be achieved with template samplesdetected within 1 × 101-1 × 106 copies. The minimum bacteriaconcentration can be detected by the assay reach 1 × 102 cfu / ml.The assay had good precision. The positive controls and negativecontrol materials measured three times at different times or at thesame time repeat the experiment five times, from which the CVvalues were calculated less than 5%. Conclusion   Thisstudy established the composite prober fluorescent quantitative PCRassay for Yersinia pestis detection, which can rapidly detectYersinia pestis bacteria and has important meaning on screening anddiagnosis of plague.

    Key words:   Composite probe;Real-time fluorescent PCR; Yersinia pestis

    《中国国境卫生检疫杂志》