Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real‐time recombinase polymerase amplification,Letters in Applied Microbiology

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Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real‐time recombinase polymerase amplification
Letters in Applied Microbiology ( IF 2.0 ) Pub Date : 2020-11-08 , DOI: 10.1111/lam.13364
Y Xu _{1,

2} , P Wu ₂ , H Zhang ₃ , J Li ₁

Affiliation

Tuberculosis (TB), as a common infectious disease, still remains the severe challenges to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying Mycobacterium tuberculosis within 20 min at 39 °C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify Mycobacterium tuberculosis with the detection limit of 4.0 copies/μL, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from Mycobacterium tuberculosis with high reliability (100%, 22/22). More important, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80.0-96.0%) and specificity (98%, 95% CI: 89.0-100.0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

中文翻译：

基于抗原 85B 的实时重组酶聚合酶扩增快速检测结核分枝杆菌

结核病作为一种常见的传染病，仍然是公共卫生面临的严峻挑战。由于目前可用的诊断工具的临床需求未得到满足，需要建立一种新的方法来普遍检测结核分枝杆菌。在此，我们设计了一种实时重组酶聚合酶扩增 (RPA) 技术，用于通过定制设计的寡核苷酸引物和探针在 39°C 下 20 分钟内识别结核分枝杆菌，该技术可以特异性靶向抗原 85B (Ag85B)。特别是，在 RPA 分析的四对设计引物中，引物 F4-R4 与探针产生最快的荧光信号。最佳引物/探针组合可有效识别结核分枝杆菌，检测限为4.0拷贝/μL，因为它不能显示来自其他分枝杆菌或病原体的基因组 DNA 的阳性信号。基于 Ag85B 的 RPA 可以确定从结核分枝杆菌中提取的基因组 DNA，可靠性高 (100%, 22/22)。更重要的是，在检测临床痰液样本时，与传统涂片相比，实时 RPA 显示出令人钦佩的灵敏度（90%、95% CI：80.0-96.0%）和特异性（98%、95% CI：89.0-100.0%）显微镜检查，类似于 Xpert MTB/RIF 的测定。这种基于实时 RPA 的 Ag85B 为快速和普遍诊断结核病提供了一种很有前景的策略。与传统涂片显微镜相比，实时 RPA 显示出令人钦佩的灵敏度 (90%, 95% CI: 80.0-96.0%) 和特异性 (98%, 95% CI: 89.0-100.0%)，类似于Xpert 山地车/RIF。这种基于实时 RPA 的 Ag85B 为快速和普遍诊断结核病提供了一种很有前景的策略。与传统涂片显微镜相比，实时 RPA 显示出令人钦佩的灵敏度 (90%, 95% CI: 80.0-96.0%) 和特异性 (98%, 95% CI: 89.0-100.0%)，类似于Xpert 山地车/RIF。这种基于实时 RPA 的 Ag85B 为快速和普遍诊断结核病提供了一种很有前景的策略。

更新日期：2020-11-08

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