Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Autor: Zhu H; Guangxi Yangxiang Co., LTD, Guigang, 537100, China; National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Wang G; Guangxi Yangxiang Co., LTD, Guigang, 537100, China., Liu X; National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, 430070, Hubei, China; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Wu W; National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, 430070, Hubei, China; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Yu T; Guangxi Yangxiang Co., LTD, Guigang, 537100, China., Zhang W; Guangxi Yangxiang Co., LTD, Guigang, 537100, China., Liu X; Guangxi Yangxiang Co., LTD, Guigang, 537100, China; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Cheng G; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Wei L; National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Ni L; Guangxi Yangxiang Co., LTD, Guigang, 537100, China., Peng Z; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Li X; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Xu D; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Qian P; National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, 430070, Hubei, China; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China., Chen P; College of Animal Science & Technology, Collegel of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. Electronic address: yxbigsuper@gmail.com.
Jazyk: angličtina
Zdroj: Microbial pathogenesis [Microb Pathog] 2024 Jun; Vol. 191, pp. 106646. Date of Electronic Publication: 2024 Apr 16.
DOI: 10.1016/j.micpath.2024.106646
Abstrakt: Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/μL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.
Competing Interests: Declaration of competing interest Hechao Zhu, Geng Wang, Teng Yu, Weichao Zhang and Lumei Ni are employees of Guangxi Yangxiang Co., LTD. The remaining authors declared that they have no competing interests.
(Copyright © 2024 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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