Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples

Nie, Min; Zhang, Ruiqing; Zhao, Mengchuan; Tan, He; Hu, Yaxin; Fan, Guohao; Li, Jingyi; He, Anna; Tian, Fengyu; Li, Feng-yu; Zheng, Ye-huan; Shen, Xinxin; Tie, Yanqing

doi:10.1016/j.mimet.2022.106504

Cited by 4 publications

(1 citation statement)

References 39 publications

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“…Attention to RAA has been increasing in recent years because of its ability to detect various pathogenic microorganisms. The numerous advantages of this technique include simple primer design, fast amplification speed, high sensitivity, low equipment requirements, no need for expensive equipment, simple operation, and intuitive result evaluation [ 41 , 42 , 43 , 44 , 45 , 46 ].…”

Section: Introductionmentioning

confidence: 99%

Rapid Onsite Visual Detection of Orf Virus Using a Recombinase-Aided Amplification Assay

Guan

Li³

et al. 2023

Life

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Orf is an important zoonotic disease caused by the Orf virus (ORFV) which can cause contagious pustular dermatitis in goats and sheep. Orf is widespread in most sheep-raising countries in the world, causing huge economic losses. Although diagnostic methods for ORFV infection already exist, it is still necessary to develop a time-saving, labor-saving, specific, low-cost and visual diagnostic method for rapid detection of ORFV in the field and application in grassroots laboratories. This study establishes a DNA extraction–free, real-time, visual recombinase–aided amplification (RAA) method for the rapid detection of ORFV. This method is specific to ORFV and does not cross-react with other common DNA viruses. The detection limits of the real-time RAA and visual judgment of the RAA assay at 95% probability were 13 and 21 copies per reaction for ORFV, respectively. Compared with qPCR, the sensitivity and specificity of the real-time RAA assay were 100%, and those of the visual RAA assay were 92.31% and 100.0%, respectively. The DNA extraction–free visual detection method of RAA established in this study can meet the needs of rapid onsite detection and grassroots laboratories and has important reference value and significance for the early diagnosis of diseased animals.

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Section: Introductionmentioning

confidence: 99%

Rapid Onsite Visual Detection of Orf Virus Using a Recombinase-Aided Amplification Assay

Guan

Li³

et al. 2023

Life

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Dye‐based recombinase‐aided amplification assay with enhanced sensitivity and specificity

Zhao,

You,

Hua

et al. 2024

iLABMED

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ObjectiveFluorescent recombinase‐aided amplification (RAA) assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity, compared with real‐time PCR (qPCR) assays, but they require a complex probe design. To eliminate the addition of fluorescent probes for RAA, an EvaGreen dye‐based recombinase‐aided amplification (EvaGreen‐RAA) assay using self‐avoiding molecular recognition system (SAMRS) primers was developed.MethodsThe SAMRS primers effectively avoided the production of primer dimers, thus improving the detection sensitivity, while EvaGreen dye was used to quantitatively measure the amplified products in real time. Using Staphylococcus aureus (SA) and Listeria monocytogenes (LM) as examples, EvaGreen‐RAA with SAMRS primers was developed. As a reference and comparison, a traditional fluorescence probe RAA method and a RAA with SAMRS primers (SAMRS‐RAA) for detecting SA and LM were also investigated. Serial dilutions of recombinant plasmids were used to evaluate the sensitivity of the assays. Unenriched and enriched simulated milk samples were used to evaluate the limits of detection (LOD) of these methods. Using high‐resolution melting (HRM) was used to explore the sensitivity of the dual EvaGreen‐RAA assay.ResultsThe sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS‐RAA and EvaGreen‐RAA for detecting SA and LM plasmids was 1 copies/μL. The LOD values of the EvaGreen‐RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL, respectively, and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL. EvaGreen‐RAA had linear amplification in real time in the range of 1–105 copies/μL of the plasmids of SA and LM. The sensitivity of the dual EvaGreen‐RAA assay for SA and LM was estimated to be 102 CFU/mL.ConclusionA real‐time quantitative EvaGreen‐RAA method for detecting SA and LM was developed, which eliminates the need to design complex RAA probes. This dye‐based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens, which has many potential applications.

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A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

Sun¹,

Lu²,

Tie³

et al. 2023

Biosafety and Health

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Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples

Cited by 4 publications

References 39 publications

Rapid Onsite Visual Detection of Orf Virus Using a Recombinase-Aided Amplification Assay

Rapid Onsite Visual Detection of Orf Virus Using a Recombinase-Aided Amplification Assay

Dye‐based recombinase‐aided amplification assay with enhanced sensitivity and specificity

A one-step reverse-transcription recombinase aided PCR assay for the rapid and sensitive detection of human enteroviruses

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