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首页> 外文期刊>Journal of Veterinary Research >A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms
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A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms

机译：一种新的，快速，简单的PMA-QPCR方法，用于检测和计数活性布鲁氏菌生物

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摘要

Introduction: The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. Material and Methods: Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. Results: The optimal exposure time and working concentration of PMA were 10 min and 15 mu g/mL, respectively. The correlation coefficient (R-2) of the standard curve was 0.999. The sensitivity of the method was 10(3) CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. Conclusion: In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.
机译：介绍：在宿主或细胞中，目前广泛使用的板计数方法从非活不带斑点中可行，是耗时和费力的。因此，有必要建立一种快速，简单的检测和计数活性布鲁氏菌生物的方法。材料和方法：使用单氮化物（PMA）来抑制来自死布氏菌的DNA的扩增，建立了一种新的，快速的PMA定量PCR（PMA-QPCR）检测方法，用于计数活性布鲁氏菌。将标准的重组质粒与插入靶BCSP31基因片段的标准重组质粒被构建用于拉伸标准曲线。优化反应条件，分析了灵敏度，特异性和可重复性。结果：PMA的最佳暴露时间和工作浓度分别为10分钟和15μg/ ml。标准曲线的相关系数（R-2）为0.999。该方法的敏感性为10（3）CFU / mL，此外，其特异性和重复性也符合要求。通过PMA-QPCR测量的B. suis的浓度与通过板计数方法测量的浓度没有显着差异，并且由两种方法测定的受感染细胞中的活细菌的浓度具有相同的数量级。结论：在这项研究中，建立了一种快速简单的PMA-QPCR计数方法，可促进相关研究。

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  • 来源
    《Journal of Veterinary Research》 |2020年第2期|共9页
  • 作者

    Zhang Shi-Jun; Wang Lu-Lu; Lu Shi-Ying; Hu Pan; Li Yan-Song; Zhang Ying; Chang Heng-Zhen; Zhai Fei-Fei; Liu Zeng-Shan; Li Zhao-Hui; Ren Hong-Lin;

  • 作者单位

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

    Jilin Univ Coll Vet Med Key Lab Zoonosis Res Minist Educ Inst Zoonosis Coll Anim Sci Xi An Da Lu 5333 Changchun 130062 Peoples R China;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 动物医学（兽医学）;
  • 关键词

    Brucella; viable bacteria; BCSP31gene; propidium monoazide; quantitative PCR;

    机译：Brucella;活细菌;BCSP31庚烯;单氮杂锭;定量PCR;

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