木糖葡萄球菌实时荧光定量 PCR 检测方法的建立及其应用
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上海实验动物研究中心,上海 201203

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Establishment of a qPCR method to detect Staphylococcus xylosus and its application
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Shanghai Laboratory Animal Research Center, Shanghai 201203, China

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    摘要:

    目的 本研究拟建立一种灵敏快速的实时荧光定量 PCR(real-time quantitative PCR, qPCR)方法,用于检测大、小鼠木糖葡萄球菌(Staphylococcus xylosus, S. xylosus)。 方法 本研究选择特异性 gehM 基因片段作为靶标合成了一套引物,建立了木糖葡萄球菌检测的 qPCR 方法。 对木糖葡萄球菌标准菌株和其他非目标菌进行特异性分析。 将木糖葡萄球菌的 DNA 进行 10 倍稀释测定其灵敏度。 用送检的样本进行了临床应用并测序验证,同时与培养法进行比较。 结果 仅木糖葡萄球菌出现特异性扩增曲线,而其他非目标菌未出现,表明设计的引物对木糖葡萄球菌具有特异性,灵敏度为 100 fg / μL,组内和组间重复性均小于 3%。 共检测 60 份临床样品,有 5 份样品扩增曲线为典型的 S 曲线,将该 qPCR 产物克隆测序并进行同源性比对,该序列与木糖葡萄球菌的同源性为 99. 63%,表明该样本木糖葡萄球菌核酸阳性,所检测样本阳性率为 8. 3%,而培养法的阳性率为 6. 7%,qPCR 方法阳性检出率比培养法略高。 结论 建立的木糖葡萄球菌 qPCR 方法具有快速、灵敏度高、特异性强和重复性好的优点,可用于实验动物木糖葡萄球菌的检测。

    Abstract:

    Objective To establish and evaluate a method for rapid and sensitive S. xylosus detection using qPCR(real-time quantitative PCR). Methods A gehM gene fragment was selected as the target for S. xylosus. A set of specific primers was synthesized and a qPCR method was established to detect S. xylosus. A S. xylosus standard strain and other non-target strains were chosen for analysis. DNA of S. xylosus was diluted 10-fold to determine its sensitivity. Clinical samples were tested, and positive products were sequenced. The result were compared with those of bacterial culture. Results S. xylosus had a specific amplification curve, whereas other non-S. xylosus species did not, indicating that the primers were specific for S. xylosus. Sensitivity was 100 fg / μL DNA. Repeatability within and between groups was less than 3%. A total of 60 clinical samples were analyzed, of which five samples had a typical S curve. qPCR products were sequenced and BLAST searched. The similarity of the gene sequences was 99. 63%, indicating that the sample was positive for the S. xylosus gehM gene with a positivity rate of 8. 3%. However, the positivity rate of bacterial culture was 6. 7%. The positivity rate of qPCR was slightly higher than that of the culture. Conclusions The established qPCR method is rapid with high sensitivity and specificity, and can be used to detect S. xylosus.

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于灵芝,冯丽萍,孔志豪,朱琦,魏晓锋.木糖葡萄球菌实时荧光定量 PCR 检测方法的建立及其应用[J].中国比较医学杂志,2024,34(01):73~79.

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  • 收稿日期:2023-06-12
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  • 在线发布日期: 2024-05-13
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自2024年1期开始,杂志参考文献改为中英文对照,具体格式要求可置下载中心查看!
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