1. Fever pathogens

1.1 Rapid visual detection of dengue virus by combining reverse transcription recombinase-aided amplification with lateral-flow dipstick assay

1.2 A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus

1.3 Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus

1.4 Rapid detection of yellow fever virus using recombinase-mediated amplification

1.5 Rapid detection of chikungunya virus using reverse transcription recombinase-mediated amplification technology

1.6 Establishment of a method for detecting Zika virus using real-time fluorescence reverse transcription recombinase-mediated isothermal amplification technology

1.7 Reverse transcription recombinase-mediated amplification detection method for West Nile virus

1.8 Establishment of a method for detecting 5 arboviruses using dual fluorescence RT-RAA

1.9 Rapid detection method for Tahyna virus using recombinase-mediated amplification

2. Infectious pathogens

2.1 A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction

2.2 Field applicable detection of hepatitis B virus using internal controlled duplex recombinase-aided amplification assay and lateral flow dipstick assay

3. Respiratory pathogens

3.1 Multiple-centre clinical evaluation of an ultrafast single-tube assay for SARS-CoV-2 RNA

3.2 A Reverse-Transcription Recombinase-Aided Amplification Assay for Rapid Detection of the 2019 Novel Coronavirus (SARS-CoV-2)

3.3 A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field

3.4 Use of a rapid reverse-transcription recombinase-aided amplification assay for respiratory syncytial virus detection

3.5 Development of a duplex reverse transcription recombinase-aided amplification assay for respiratory syncytial virus incorporating an internal control

3.6 A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling

3.7 Use of a rapid recombinase-aided amplification assay for Mycoplasma pneumoniae detection

3.8 Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7

3.9 Establishment of a recombinase-mediated nucleic acid detection method for Middle East respiratory syndrome coronavirus

3.10 Establishment and application of a rapid detection method for Middle East respiratory syndrome coronavirus RT_RAA

3.11 Research on a rapid detection method for influenza A virus using reverse transcription recombinase-mediated nucleic acid amplification

3.12 Methodological research on rapid detection of human rhinovirus using recombinase-mediated isothermal amplification

3.13 Patent information analysis of nucleic acid detection and diagnosis for novel coronavirus pneumonia

3.14 Establishment and evaluation of a novel coronavirus nucleic acid fluorescence RT-RAA detection method

3.15 Research on rapid detection of respiratory adenovirus based on RAA fluorescence method

4. Mycobacterium pathogens

4.1 Application of recombinase-mediated isothermal amplification method to establish a method for detecting non-tuberculous mycobacteria

4.2 Application value of probe-guided recombinase-mediated isothermal amplification method for detecting TB_rpoB gene mutation

4.3 The value of rapid diagnosis of sputum smear-negative pulmonary tuberculosis by combining real-time detection technology of RNA isothermal amplification with fluorescence quantitative PCR

5. Intestinal pathogens

5.1 Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

5.2 Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA

5.3 RAA combined with CRISPR-Cas13a for rapid detection of 4 kinds of diarrheal pathogens

6. Disease surveillance

6.1 Development and evaluation of a rapid detection assay for severe fever with thrombocytopenia syndrome virus based on reverse-transcription recombinase polymerase amplification

6.2 Integrating Microwave Resonator and Microchannel with Immunochromatographic Strip for Stable and Quantitative Biodetection

6.3 Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent

6.4 A probe directed recombinase amplification assay for detection of MTHFR A1298C polymorphism associated with congenital heart disease

Rapid visual detection of dengue virus by combining reverse transcription recombinase-aided amplification with lateral-flow dipstick assay

Abstract: Objectives: Dengue, caused by infection with the dengue virus (DENV), is endemic in tropical and subtropical regions worldwide and is a major public health concern. With more large outbreaks occurring in rural areas, this study aimed to develop a point-of-care test using recombinase-aided amplification and lateral-flow dipsticks for rapid DENV detection in low-resource settings. Methods: Primers for the recombinase-aided amplification (RAA) assay were designed based on the 3'UTR of the DENV genome and screened. The RAA temperature, time, and primer concentration were optimized, as was the lateral-flow dipstick assay (LFD) time. Finally, the diagnostic performance of the reverse transcription (RT)-RAA-LFD assay was evaluated using blood samples from 247 patients clinically suspected of DENV infection. Results: The RAA primer pair F1/R2 was optimal for detecting DENV. The RT-RAA was performed in an incubator block at 37℃ for 20 minutes, and amplicons were visible on the flow dipsticks to the naked eye within 3 minutes. The detection limit of the developed RT-RAA-LFD assay was 10 copies/mL with high specificity for DENV. Compared with a commercial reverse transcription quantitative PCR assay, the kappa value of RT-RAA-LFD in the 247 clinical samples was 0.957. Conclusions: This study developed a rapid and visual point-of-care test based on RT-RAA and LFD assay. It proved suitable for reliable DENV detection in low-resource settings with limited laboratory capabilities and optimal storage conditions.

Keywords: Dengue virus, Recombinase-aided amplification, Lateral-flow dipstick assay.

A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus

Abstract: Objective: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. Methods: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 ℃ within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. Results: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. Conclusion: A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.

Key words: Tick-borne encephalitis virus; Subtype; Far-eastern; Detection; RT-RAA.

Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus

West Nile virus (WNV) causes West Nile fever and West Nile encephalitis. Because infection by WNV creates serious public health problems, its simple, rapid, and visual detection is very important in clinical practice, especially in resource-limited laboratories. We have developed a rapid, specific, and highly sensitive internally controlled reverse transcription recombinase-aided amplification (RT-RAA) assay to detect WNV, using both real-time fluorescence and the lateral flow dipstick (LFD) at 39 ℃ for 30 min. The analytical sensitivity of the RT-RAA assay was 10 plasmid copies and 1.6 pfu per reaction with real-time fluorescence, and 100 plasmid copies per reaction with the LFD. No cross-reaction with other control viruses was observed. Compared with the RT-qPCR assay, the RT-RAA assay demonstrated 100% sensitivity and 100% specificity for WNV.

Key words: WNV; Detection; RT-RAA; Lateral flow dipstick (LFD).

Recombinase-mediated amplification method for rapid detection of yellow fever virus

Abstract: Objective: This study uses the recombinase-mediated isothermal nucleic acid amplification method (RAA) to establish a one-step isothermal nucleic acid amplification (RT-RAA) method for yellow fever virus by using reverse transcriptase. Methods: Primers and probes were designed based on the conserved sequences of the yellow fever virus genome, and the reproducibility, specificity, and sensitivity of RT-RAA were established and analyzed; therefore, the established method was used to detect yellow fever virus samples, and gene sequencing was used for verification. Results: In the yellow fever virus RT-RAA amplification, the addition of 40U of reverse transcriptase produced the best amplification effect. This method has a short detection time (<20min) and high sensitivity, with a detection limit of 100 copies. There was no cross-reaction with mosquito-borne viruses such as dengue virus, West Nile virus, Japanese encephalitis virus, and chikungunya virus, indicating good specificity. Conclusion: The constructed RT-RAA method for yellow fever virus is rapid, specific, and sensitive, suitable for rapid detection of yellow fever virus at ports of entry.

Keywords: Recombinase-mediated amplification; Yellow fever virus; Molecular detection.