1. Pet pathogens

1.1 Establishment of a fluorescence RT-RAA detection method for feline coronavirus

1.2 Research on Brucella detection methods and kits based on isothermal amplification technology

2. Poultry diseases

2.1 Establishment of reverse transcription recombinase-aided amplification-lateral-flow dipstick and real-time fluorescence-based reverse transcription recombinase-aided amplification methods for detection of the Newcastle disease virus in chickens

2.2 RAA-LFD assay - a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus

2.3 Reverse transcription recombinase-aided amplification assay combined with a lateral flow dipstick for detection of avian infectious bronchitis virus

2.4 Research Note: Rapid detection of avian infectious laryngotracheitis virus with real-time fluorescence-based recombinase-aided amplification

2.5 Reverse-transcription recombinase-aided amplification assay for H7 subtype avian influenza virus

2.6 Detection of H7N9 avian influenza virus by recombinase-mediated isothermal amplification

2.7 Establishment of a visual detection method for avian influenza RT-RAA lateral flow test strips

2.8 Establishment of a rapid fluorescence RAA detection method for Pasteurella multocida

2.9 Recombinase-mediated detection method study of avian influenza virus H5N1

3. Animal diseases

3.1 Clinical Validation of Two Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of African Swine Fever Virus

3.2 Establishment of a real-time fluorescence RAA detection method for African swine fever virus

3.3 Establishment of a fluorescence detection method for porcine erythrocyte-associated recombinase-mediated isothermal amplification technology RAA

3.4 Establishment of a rapid diagnostic method for bovine viral diarrhea virus type 1 RT-RAA

3.5 Establishment of a real-time fluorescence RAA detection method for porcine circovirus type 4

3.6 Establishment of a recombinase-mediated fluorescence nucleic acid amplification detection system for Sclerotinia sclerotiorum

4. Plant diseases

4.1 CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus

Establishment of a fluorescence RT-RAA detection method for feline coronavirus

Abstract: Based on the 7b gene sequence of feline coronavirus (FCoV), primers and probes were designed to establish a fluorescence RT-RAA detection method for FCoV. This method can rapidly detect pathogens within 20 min at a constant temperature of 39 ℃, and there is no cross-reaction with feline herpesvirus, feline calicivirus, and feline parvovirus; the minimum detection limit for viral nucleic acid is 1.97 × 101 fg/uL. Thirty suspected FCoV-infected clinical samples were detected using nested PCR and the established fluorescence RT-RAA, and the coincidence rate of the two methods was 93.33%. The results show that the established FCoV fluorescence RT-RAA method is suitable for rapid detection of FCoV.

Keywords: Feline coronavirus; Fluorescence RT-RAA; Rapid detection.

Research on Brucella detection methods and kits based on isothermal amplification technology

Abstract: This paper introduces the harm and detection technology of brucellosis, studies the isothermal amplification technology of Brucella, and illustrates that it can be used to detect samples with low Brucella DNA content. The developed kit can perform isothermal amplification detection at 30 ℃～42 ℃, and the detection can be completed in 5～20 min. It points out that the kit has the advantages of being fast, sensitive, accurate, and reproducible, suitable for grassroots and on-site detection, and has good application prospects.

Keywords: Isothermal amplification; Brucella; Detection method; Kit.

Establishment of reverse transcription recombinase-aided amplification-lateral-flow dipstick and real-time fluorescence-based reverse transcription recombinase-aided amplification methods for detection of the Newcastle disease virus in chickens

ABSTRACT Newcastle disease is an acute and highly contagious disease of poultry caused by Newcastle disease virus infection, which does great harm to the poultry industry all over the world. To diagnose the disease simply and quickly, 2 detection methods were established based on reverse transcription recombinase-aided amplification (RT-RAA) technology. One is reverse transcription recombinase-aided amplification lateral flow dipstick (RT-RAA-LFD) that is to combine RT-RAA with lateral flow dipstick; the other is real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) that is the combination of RT-RAA and exo probe. In this study, the reaction conditions such as reaction temperature and reaction time of the 2 methods were optimized, and their specificity and sensitivity were tested. The results showed that the RT-RAA-LFD method could be used to complete reaction within 23 min, and its lowest detectable limit was 102 copies/mL, 10 times higher than that of the conventional PCR method (103 copies/mL); the RF-RT-RAA method could be used to complete reaction within 26 min, and its lowest detectable limit was 10 copies/mL, 100 times higher than that of conventional PCR method (103 copies/mL), and it was as sensitive as real-time fluorescence-based quantitative PCR (10 copies/mL). The 2 methods had no cross-reaction to the nucleic acid of other avian pathogens and showed good specificity. A total of 86 clinical samples suspected of the Newcastle disease virus were tested by conventional PCR, real-time fluorescence-based quantitative PCR, RT-RAA-LFD, and RF-RT-RAA. Based on the commonly used conventional PCR method, the other 3 detection methods had a coincidence rate of higher than 93%. In summary, RT-RAA-LFD and RF-RT-RAA had high specificity, sensitivity, and efficiency, which were suitable for clinical and laboratory diagnosis, respectively, and provided technical support for the prevention and control of Newcastle disease.

Key words: Newcastle disease virus, reverse transcription recombinase-aided amplification-lateral flow dipstick, real-time fluorescence-based-reverse transcription recombinase-aided amplification, detection.

RAA-LFD assay - a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus

ABSTRACT: This study aimed to develop a specific, simple, and sensitive method for diagnosing avian infectious laryngotracheitis virus (ILTV). Recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) were combined, labeling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the 5' end of the downstream primer with biotin. By optimizing the RAA reaction time, temperature, and primer concentration, an RAA-LFD assay for detecting ILT was established. Specificity and sensitivity testing showed that the target gene fragments could be amplified by the RAA-LFD assay in 20 minutes under isothermal conditions (37°C), and amplification products could be visually observed and determined by LFD within 3 minutes. No cross-reaction with nucleic acids of other avian pathogens was observed. The lowest detectable limit (LDL) of the RAA-LFD assay was 10² copies/µL, 100 times higher sensitivity than conventional CR with an LDL of 10⁴ copies/µL. The RAA-LFD assay proved highly sensitive, easy to use, and suitable for clinical detection.

Key words: avian infectious laryngotracheitis virus; recombinase-aided amplification; lateral flow dipstick