• 中文核心期刊
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灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立

SYBR Green I RT-PCR Assay for Quantitative Detection of Salmonella in Irrigation Water

    摘要
  • 摘要:
      目的  建立基于SYBR Green I嵌合染料法的灌溉水源中沙门氏菌实时荧光定量PCR（Quantitative Real-time PCR, qPCR）检测方法。
      方法  以沙门氏菌侵袭蛋白A （invA）基因为靶基因，设计1对特异性qPCR检测引物，在对qPCR反应体系和反应条件进行优化后，通过特异性实验和灵敏度实验对qPCR方法进行验证，并制作灌溉水源中沙门氏菌qPCR定量标准曲线，最后与国标方法对比检测灌溉水源中沙门氏菌污染来验证该方法的准确性。
      结果  经过优化的qPCR检测方法灵敏度检测结果显示最低检测限量为1×10−1 pg·μL−1，特异性检测结果显示具有良好的特异性，干扰菌株对本qPCR检测方法无影响，所制作的qPCR扩增标准曲线在2×100～2×105 cfu·mL−1有较好的线性关系，相关系数为0.999 6，能对沙门氏菌进行准确的定量分析。该方法在与国标方法对比检测灌溉水源中沙门氏菌污染时具有同等准确性。
      结论  本研究建立的基于SYBR Green I嵌合荧光法的实时荧光定量PCR检测方法，可以满足灌溉水源中沙门氏菌的定量检测要求。

     

    Abstract:
      Objective  A SYBR Green I-based quantitative RT-PCR (qPCR) method was developed for detecting salmonella in irrigation water.
      Method   For the methodology development a pair of primers were designed based on the sequences of the invasion protein A gene (invA) of Salmonella. qPCR reaction conditions were optimized, the assay tested for specificity and sensitivity, and a standard curve of amplification constructed. Test results on a specimen of contaminated irrigation water using the qPCR assay were compared for detection accuracy with those obtained from the national standard method.
      Result  The newly developed qPCR assay showed a minimum detection limit of 1×10−1pg·μL−1 and free of interference from genomic nucleic acids of non-target microbes. The constructed linear standard curve between 2×100cfu·mL−1 and 2×105 cfu·mL−1 had a high correlation coefficient of 0.999 6. The assay demonstrated same accuracy as did the national standard method in detecting salmonella in irrigation water.
      Conclusion  The newly established qPCR assay could be adequately applied for salmonella detection in agriculture irrigation water.

     

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