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首页> 外文期刊>RSC Advances >Establishment of a rapid and sensitive method based on recombinase polymerase amplification to detect mts90, a new molecular target of Mycobacterium tuberculosis
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Establishment of a rapid and sensitive method based on recombinase polymerase amplification to detect mts90, a new molecular target of Mycobacterium tuberculosis

机译：基于重组酶聚合酶扩增的快速和敏感方法的建立检测MTS90，结核分枝杆菌的新分子靶标

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摘要

Tuberculosis (TB) remains a significant challenge to public health, especially in developing countries. Failure in early diagnosis and lack of rapid and accurate diagnostic methods lead to ongoing prevalence and transmission of TB. Recently, the recombinase polymerase amplification (RPA) technique has made it possible to rapidly amplify and detect nucleic acids without specialized devices. We developed a RPA-based method for identifying Mycobacterium tuberculosis (MTB) by detecting mts90, a more specific target identified in our previous research. Different screening methods were employed for selecting a preferred primer pair of amplification, and probes were confirmed as very fast and reliable tools in the screening of potential primer candidates. The results showed that the mts90 RPA assay was very sensitive and capable of detecting 6 copies of recombinant plasmid containing mts90 sequence per reaction. The assay was specific for detecting MTB, as it did not identify the genomic DNA from other mycobacteria and pathogens. When applied to analyze clinical samples, including sputum, bronchoalveolar lavage fluid (BALF) and tissues, the mts90 RPA assay had a coincidence rate of 96.43% (27/28) compared to the Biochip test, which has been used in clinics for diagnosing TB. The mts90 RPA assay can be completed within 20 minutes at 39 degrees C without thermal cycling; its simple operation and rapid detection suggest RPA-based MTB assays could be further developed for TB diagnosis in resource-poor settings.
机译：结核病（TB）对公共卫生仍然是一个重大挑战，特别是在发展中国家。早期诊断失败，缺乏快速和准确的诊断方法导致TB的持续流行和传播。最近，重组酶聚合酶扩增（RPA）技术使得可以快速扩增和检测没有专门器件的核酸。我们开发了一种基于RPA的方法，用于通过检测MTS90来鉴定结核分枝杆菌（MTB），在我们以前的研究中发现的更具体的目标。使用不同的筛选方法来选择优选的引物对扩增，并且在筛选潜在的引物候选物中被证实了探针是非常快速可靠的工具。结果表明，MTS90 RPA测定非常敏感，能够检测每次反应含有MTS90序列的重组质粒的6份。测定对于检测MTB是特异性的，因为它没有鉴定来自其他分枝杆菌和病原体的基因组DNA。当应用于分析临床样品时，包括痰，支气管肺泡灌洗液（BALF）和组织，与Biochip试验相比，MTS90 RPA测定的重合率为96.43％（27/28），这已被用于诊断结核病的诊所。 MTS90 RPA测定可以在39℃下在没有热循环的情况下在20分钟内完成;其简单的操作和快速检测表明基于RPA的MTB测定可以进一步开发用于资源差的设置中的结核病诊断。

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  • 来源
    《RSC Advances》 |2017年第79期|共8页
  • 作者

    Mo Yunjun; Cui Fang; Li Dairong; Dai Yi; Li Xinmin; Zhang Xinyuan; Qiu Yulan; Yin Yibing; Zhang Xuemei; Xu Wenchun;

  • 作者单位

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

    Chongqing Med Univ Affiliated Hosp 1 Dept Lab Med Chongqing 400016 Peoples R China;

    Chongqing Med Univ Affiliated Hosp 1 Dept Resp Med Chongqing 400016 Peoples R China;

    Chongqing Med Univ Inst Life Sci Chongqing 400016 Peoples R China;

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

    Chongqing Med Univ Sch Lab Med Minist Educ Key Lab Lab Med Diagnost Designated 1 Yixueyuan Rd Chongqing 400016 Peoples R China;

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