Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2

Research (Published online: 19-11-2023)

18. Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2

Ploypassorn Homklinkaew, Sakuna Phatthanakunanan, Siriluk Jala, Alongkot Boonsoongnern, and Preeda Lertwatcharasarakul

Veterinary World, 16(11): 2313-2320

Ploypassorn Homklinkaew: Animal Health and Biomedical Sciences Study Program, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.

Sakuna Phatthanakunanan: Kamphaeng Saen Veterinary Diagnostic Center, Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom 73140, Thailand.

Siriluk Jala: Kamphaeng Saen Veterinary Diagnostic Center, Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom 73140, Thailand.

Alongkot Boonsoongnern: Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom 73140, Thailand.

Preeda Lertwatcharasarakul: Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Nakorn Pathom 73140, Thailand.

Background and Aim: Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples.

Materials and Methods: To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR.

Results: The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5’ end of the forward primer and with biotin at the 5’ end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/μL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen’s kappa coefficients showed a good agreement with the established techniques.

Conclusion: The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field.

Keywords: lateral flow dipstick assay, Porcine circovirus type 2, recombinase-aided amplification.