Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus.

Autor: Ye H; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Wang X; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety, Beijing Laboratory for Food Quality and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Zhou L; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Ge X; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Gao P; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Han J; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Guo X; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Wen K; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Beijing Key Laboratory of Detection Technology for Animal Derived Food Safety, Beijing Laboratory for Food Quality and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China., Zhang Y; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China. Electronic address: zhangyongning@cau.edu.cn., Yang H; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
Jazyk: angličtina
Zdroj: Microbial pathogenesis [Microb Pathog] 2024 Oct; Vol. 195, pp. 106885. Date of Electronic Publication: 2024 Aug 24.
DOI: 10.1016/j.micpath.2024.106885
Abstrakt: Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 10 0 TCID 50 PEDV, 1 × 10 4 TCID 50 PDCoV, and 1 × 10 2 TCID 50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.
Competing Interests: Declaration of competing interest The authors declare that they have no competing interests.
(Copyright © 2024 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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