Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing <i>Escherichia coli</i>

Cao, Yuhao; Fang, Taisong; Shen, Jinling; Zhang, Guodong; Guo, Dehua; Zhao, Lina; Yuan, Jun; Zhi, Shuai; Zheng, Lin; Lv, Xiaofei; Yao, Zhiyuan; Yu, Daniel

doi:10.1093/jaoacint/qsad063

Cited by 6 publications

(3 citation statements)

References 24 publications

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“…However, real-time PCR requires varying cycling temperatures at different steps, rigorous conditions, and a run time of at least 1 h. RAA assays have been applied to detect many pathogens, including mpox virus and M. pneumoniae. ,− Compared with real-time PCR, RAA has several advantages, including complete detection under simple reaction conditions including a constant temperature (∼39 °C) and a shorter processing time (20 min in total). LAMP, another rapid detection assay using isothermal amplification, has also been developed to detect C.…”

Section: Discussionmentioning

confidence: 99%

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris

Feng,

Chen,

et al. 2024

Anal. Chem.

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Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay’s efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/μL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102–103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.

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Section: Discussionmentioning

confidence: 99%

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris

Feng,

Chen,

et al. 2024

Anal. Chem.

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“…This method offers a significant advantage as it does not require specialized PCR thermocycling equipment and can be completed in 30 min at a consistently low temperature (37-42 • C). The technique is becoming increasingly important in the discovery of bacteria and viruses and has been extensively applied in research and disease management, showing potential for in situ detection [35,36].…”

Section: Introductionmentioning

confidence: 99%

Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi

Zu,

Sun,

et al. 2024

Microorganisms

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Streptococcus equi subspecies equi (S. equi) is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the eqbE gene, for the rapid detection of S. equi nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the eqbE gene, enhancing the overall specificity and sensitivity of the detection. After efficiency optimization, this real-time RAA method can detect 10 or more copies of nucleic acid within 20 min. The method demonstrates high specificity for S. equi and does not cross-react with other clinically relevant pathogens. Real-time RAA diagnostic performance was evaluated using 98 nasal swab samples collected from horses and compared with the real-time PCR detection method. Results revealed that 64 and 65 samples tested positive for S. equi using real-time RAA and real-time PCR, respectively. The overall agreement between the two assays was 96.94% (95/98), with a kappa value of 0.931 (p < 0.001). Further linear regression analysis indicated a significant correlation in the detection results between the two methods (R2 = 0.9012, p < 0.0001), suggesting that the real-time RAA assay exhibits a detection performance comparable to that of real-time PCR. In conclusion, the real-time RAA assay developed here serves as a highly specific and reliable diagnostic tool for the detection of S. equi in equine samples, offering a potential alternative to real-time PCR methods. In conclusion, the real-time RAA nucleic acid diagnostic method, based on the eqbE gene, offers rapid and accurate diagnosis of S. equi, with the added advantage of minimal equipment requirements, thus contributing to the efficient detection of strangles in horses.

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“…There are numerous methods used to detect Shiga toxins and/or the presence of Stx genes including PCR, ELISA, LC-MS, mouse bioassays, cell-free assays, cell-based assays, biosensors, etc. [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Each of these detection assays have their own advantages and disadvantages.…”

Section: Introductionmentioning

confidence: 99%

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli

Tam,

Wang,

et al. 2024

Toxins

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Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1–0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.

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Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing Escherichia coli

Cited by 6 publications

References 24 publications

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris

Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli

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