Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Abstract

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge 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 M. 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 M. tuberculosis with the detection limit of 4·0 copies per 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 M. tuberculosis with high reliability (100%, 22/22). More importantly, 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.

Establishment and application of a rapid molecular diagnostic platform for the isothermal visual amplification of group B Streptococcus based on recombinase polymerase

“…Considering the multiple serotypes of GBS, the antigen-antibody test is prone to the risk of being false negative, leading this study to focus on the molecular diagnosis. RPA technology, with proven sensitivity and simplicity comparable with traditional PCR, has been applied diagnosing many diseases, including foot-and-mouth disease virus (Abd El Wahed et al, 2013), Dengue virus (Abd El Wahed et al, 2015), and Mycobacterium tuberculosis (Xu et al, 2021). While Yu et al…”

Rapid Detection of Klebsiella pneumoniae Carrying Virulence Gene rmpA2 by Recombinase Polymerase Amplification Combined With Lateral Flow Strips

“…Combining the isothermal RPA reaction with the LFS endpoint reading method, with short reaction times, low isothermal conditions, simple operation, and no instrumentation, offers a promising solution ( Wu et al., 2016 ). This RPA-LFS technique has been successfully applied in the molecular diagnosis of diseases caused by methicillin-resistant Staphylococcus aureus , Mycobacterium tuberculosis , Cryptococcus novelis , and other pathogenic bacteria with exploratory significance ( Yongkiettrakul et al., 2017 ; Ma et al., 2019 ; Xu et al., 2021 ) However, the technique places high demands on primer design, and a very small number of primer dimers may lead to false positives.…”

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples

“…Typically, amplifying nucleic acids can be completed under 37–42℃ condition within 20 min (McQuillan and Wilson 2021 ; Zhang et al 2021a ; Zheng et al 2021 ). Fluorescent-based and lateral flow dipstick (LFD)-based detection has been widely established for various targets among the detection format of RPA amplicons (Behrmann et al 2020 ; Shelite et al 2021 ; Wang et al 2022 ; Xu et al 2021 ). Herein, to meet the needs for rapid detection on first aid and emergency treatment, especially for resource-limited settings and poorly equipped laboratories, combining RPA assays and LFD strips (designated as RPA-LFD) is a desirable option.…”