Chines Journal of Vector Biology and Control ›› 2020, Vol. 31 ›› Issue (6): 695-698.DOI: 10.11853/j.issn.1003.8280.2020.06.014

• Investigation • Previous Articles     Next Articles

Application of SYBR GreenⅠfluorescence quantitative PCR in the rapid detection of Brucella

ZHANG Ai-ping, MA Li, XIE Hui, YANG Xu-xin, XUE Hong-mei, ZHAO Zhi-jun, LI Ji-quan, YU Shou-hong, ZHAO Zhong-zhi, XU Li-qing   

  1. Qinghai Institute for Endemic Disease Prevention and Control, Xining 810021, Qinghai Province, China
  • Received:2020-06-19 Online:2020-12-20 Published:2020-12-20
  • Supported by:
    Supported by the National Natural Science Foundation of China (No. 81860588) and the Planning Guidance Project of Qinghai Provincial Health and Family Planning Commission (No. 2018-wjzdx-84)

SYBR GreenⅠ荧光定量PCR在布鲁氏菌快速检测中的应用研究

张爱萍, 马丽, 谢辉, 杨旭欣, 薛红梅, 赵志军, 李积权, 于守鸿, 赵忠智, 徐立青   

  1. 青海省地方病预防控制所鼠疫预防控制科/布鲁氏菌病预防控制科/地方病预防控制科, 青海 西宁 810021
  • 通讯作者: 徐立青,
  • 作者简介:张爱萍,女,主管医师,主要从事鼠疫病原学检测与研究工作,
  • 基金资助:

Abstract: Objective To investigate the application of SYBR GreenⅠ fluorescence quantitative PCR in the rapid detection of Brucella, and to provide a new method for the early detection of brucellosis. Methods Bacterial isolation and culture were performed for two whole blood samples collected from patients with acute brucellosis, 8 whole blood samples collected from patients with chronic brucellosis, and 12 whole blood samples collected from sheep, and suspected Brucella strains were screened by smear microscopy and bacteriophage lysis test. Nucleic acid was extracted, templates were prepared, and then SYBR GreenⅠ fluorescence quantitative PCR was used for the detection of Brucella. Results A total of four suspected Brucella strains were isolated, with the same morphology and staining characteristics as Gram-negative bacilli. The bacteriophage lysis test for strains 1, 2, and 3 showed that BK2 lysed and Tb was not and these three strains were determined as Brucella melitensis; the bacteriophage lysis test for strain 4 yielded negative results. All strains except the strain 4 and EV76 showed a specific absorption peak at 88℃ in the melting curve of fluorescent quantitative PCR, and thus they were confirmed as Brucella. Conclusion SYBR GreenⅠ fluorescence quantitative PCR has the advantages of short time, strong specificity, and good repeatability in the detection of Brucella, and therefore, it holds promise for application in the screening for brucellosis and the rapid detection of Brucella.

Key words: SYBR Green I fluorescence quantitative PCR, Brucella, Rapid detection

摘要: 目的 将SYBR GreenⅠ荧光定量PCR技术应用于布鲁氏菌的快速检测,为布鲁氏菌病(布病)的早期发现提供新的检测手段。方法 将收集的布病急性期患者全血2份,慢性期患者全血8份和羊全血12份进行细菌分离培养,筛选疑似布鲁氏菌进行涂片镜检、噬菌体裂解试验,同时提取核酸,制备模板,利用SYBR GreenⅠ荧光定量PCR技术进行布鲁氏菌检测。结果 共分离出4株疑似布鲁氏菌,其形态及染色特征与革兰阴性小杆菌相同。1、2、3号菌株噬菌体裂解试验BK2裂解,Tb不裂解,确定为羊种布鲁氏菌,4号噬菌体裂解试验阴性。除4号待测菌株和阴性对照EV76外,其他菌株的荧光定量PCR的熔解曲线在88℃左右出现特异性的吸收峰,证实是布鲁氏菌。结论 SYBR GreenⅠ荧光定量PCR技术检测布鲁氏菌用时短,特异性强,重复性好,在布病的筛查和布鲁氏菌的快速检测中具有较好的推广应用价值。

关键词: SYBR Green Ⅰ荧光定量PCR, 布鲁氏菌, 快速检测

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