生物学杂志

生物学杂志 ›› 2025, Vol. 42 ›› Issue (2): 107-.doi: 10.3969/j.issn.2095-1736.2025.02.107

• 技术方法 • 上一篇    下一篇

多色熔解曲线法快速检测5种小鼠病原体

徐逸梦， 刘欣怡， 胡珊珊， 周宇荀， 李　凯， 肖君华   

  1. 东华大学 生物与医学工程学院， 上海 201620
  • 出版日期:2025-04-18 发布日期:2025-04-14
  • 通讯作者: 肖君华，教授，研究方向为分子遗传学，E-mail:xiaojunhua@dhu.edu.cn
  • 作者简介:徐逸梦，硕士研究生，研究方向为分子检测，E-mail:xymkackac@163.com
  • 基金资助:
    国家重点研发计划项目(2018YFA0801101)

Rapid detection of five mouse pathogens by multicolor melting curve analysis

XU Yimeng, LIU Xinyi, HU Shanshan, ZHOU Yuxun, LI Kai, XIAO Junhua   

  1. College of Biological Science and Medical Engineering, Donghua University, Shanghai 201620, China
  • Online:2025-04-18 Published:2025-04-14

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摘要： 借助多色熔解曲线分析技术（multicolor melting curve analysis, MMCA），建立起一套高效、低成本的同时检测5种小鼠病原体（金黄色葡萄球、铜绿假单胞菌、肺炎克雷伯菌、嗜肺巴斯德杆菌、变形杆菌）的多重检测方案，用于快速检测实验动物感染情况。同时添加辣椒基因组作为内参，排除假阴性干扰，使结果更准确可靠。通过对探针进行设计，使不同病原菌在不同荧光通道的不同的Tm值处出现熔解峰：在FAM荧光通道，金黄色葡萄球菌和肺炎克雷伯菌的熔解峰Tm值分别为（53.51±0.94） ℃、（61.23±0.34） ℃；在ROX通道，铜绿假单胞菌和嗜肺巴斯德杆菌的熔解峰Tm值分别为（61.70±0.66）℃、（55.98±0.10）℃；在CY5通道，变形杆菌和辣椒内参的熔解峰Tm值分别为（63.91±0.19）℃、（57.43±0.31）℃。经验证，该方法对不同病原体具有不同的检测限，分别为金黄色葡萄球菌102copies/μL、肺炎克雷伯菌101copies/μL、铜绿假单胞菌102copies/μL、嗜肺巴斯德杆菌102copies/μL、变形杆菌102copies/μL。综上，研究建立的多色熔解曲线方法能够对多种小鼠常见病原体在单管内进行快速有效的检测。

关键词: 多色熔解曲线分析, 多重PCR, 小鼠病原体, 分子检测, 快速检测

Abstract: Based on multicolor melting curve analysis, an efficient and low-cost simultaneous detection protocol of five mouse pathogens for the rapid detection of infection in experimental animals (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Pasteurella pneumotropica,andProteus) was established. Meanwhile, the capsicum genome was added as an internal reference to exclude false negatives and make the results more accurate and reliable. By designing the probes, the melting peaks of different pathogens appeared at different Tm values in different fluorescence channels: in the FAM fluorescence channel, the melting peak Tm values ofS. aureusandK. pneumoniaewere in the range of （53.51±0.94） ℃ and （61.23±0.34） ℃, respectively; in the ROX channel, the melting peaks ofP. aeruginosaandP. pneumotropicawere in the range of （61.70±0.66） ℃, （55.98±0.10） ℃; in CY5 channel, the melting peak Tm values ofProteusandCapsicum annuumL. were at （63.91±0.19） ℃, （57.43±0.31） ℃, respectively. The method was verified to have different detection limits for different pathogens with 102copies forS. aureus, 101copies forK. pneumoniae, 102copies forP. aeruginosa, 102copies forP. pneumotropica, and 102copies forProteus. In summary, the multicolor melting curve method established in this work was capable of rapid and effective detection of a wide range of common mouse pathogens in a single tube.

Key words: multicolor melting curve analysis, multiple PCR, mouse pathogens, molecular detection, rapid detection

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