Jun Tu, Zhengdan Lin, Erchao Sun, Teng Yu, Weichao Zhang, Yumei Sun, Hechao Zhu, Pin Qian, Guofu Cheng
{"title":"用于区分 PEDV、TGEV 和 PKV 的三重实时反转录聚合酶链反应测定的建立和应用","authors":"Jun Tu, Zhengdan Lin, Erchao Sun, Teng Yu, Weichao Zhang, Yumei Sun, Hechao Zhu, Pin Qian, Guofu Cheng","doi":"10.3390/vetsci11090413","DOIUrl":null,"url":null,"abstract":"<p><p>The pathogens responsible for porcine viral diarrhea are diverse, causing significant economic losses to the pig industry. PEDV and TGEV are well-known pathogens causing diarrheal diseases in pigs, leading to significant economic losses in the breeding industry. In contrast, the newly identified diarrhea virus, PKV, has not garnered as much attention. However, co-infection of PKV with PEDV results in more severe symptoms in piglets, such as acute gastroenteritis, and promotes increased replication of PEDV. Rapid and accurate diagnosis of viral diarrhea is essential for farms to identify pathogens early and mitigate economic losses. This study describes the development of a triplex real-time fluorescent quantitative RT-qPCR technique that can simultaneously detect three RNA viruses associated with porcine viral diarrhea: PEDV, TGEV, and PKV. To establish the triplex RT-qPCR method for the simultaneous detection and identification of the above three diarrhea viruses, conserved regions of the M gene of TGEV, the N gene of PEDV, and the 3D gene of PKV were selected to design specific primers and probes. After optimizing the reaction conditions, the method's specificity, sensitivity, and reproducibility were evaluated. The triplex RT-qPCR method did not show a significant difference in PCR efficiency compared to the single RT-qPCR method. The method is specific to TGEV, PKV, and PEDV, exhibits no cross-reactivity with other pathogens, and demonstrates satisfactory sensitivity and reproducibility; the limit of detection (LOD) of PEDV, TGEV, and PKV is 11.42 copies/μL. Furthermore, the performance of the triplex RT-qPCR assay was compared with the Chinese standard single-assay method for detecting TGEV, PKV, and PEDV, showing complete consistency between the two methods (100% compliant). Subsequently, 1502 clinical diarrhea samples were collected from the Guangxi Zhuang Autonomous Region to investigate the local prevalence of TGEV, PKV, and PEDV and the positive rates were 16.38% (246/1502), 1.46% (22/1502), and 45.14% (678/1502), respectively. Co-infection of PEDV and PKV were most common, with a rate of 12.12% (182/1502). This study presents a valuable method for the rapid and simultaneous identification of PEDV, TGEV, and PKV in clinical animal farming practices, and provides a reassessment of the epidemiology of these diarrhea-causing viral pathogens in the Guangxi Zhuang Autonomous Region.</p>","PeriodicalId":23694,"journal":{"name":"Veterinary Sciences","volume":null,"pages":null},"PeriodicalIF":2.0000,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11435592/pdf/","citationCount":"0","resultStr":"{\"title\":\"Establishment and Application of a Triplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of PEDV, TGEV and PKV.\",\"authors\":\"Jun Tu, Zhengdan Lin, Erchao Sun, Teng Yu, Weichao Zhang, Yumei Sun, Hechao Zhu, Pin Qian, Guofu Cheng\",\"doi\":\"10.3390/vetsci11090413\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The pathogens responsible for porcine viral diarrhea are diverse, causing significant economic losses to the pig industry. PEDV and TGEV are well-known pathogens causing diarrheal diseases in pigs, leading to significant economic losses in the breeding industry. In contrast, the newly identified diarrhea virus, PKV, has not garnered as much attention. However, co-infection of PKV with PEDV results in more severe symptoms in piglets, such as acute gastroenteritis, and promotes increased replication of PEDV. Rapid and accurate diagnosis of viral diarrhea is essential for farms to identify pathogens early and mitigate economic losses. This study describes the development of a triplex real-time fluorescent quantitative RT-qPCR technique that can simultaneously detect three RNA viruses associated with porcine viral diarrhea: PEDV, TGEV, and PKV. To establish the triplex RT-qPCR method for the simultaneous detection and identification of the above three diarrhea viruses, conserved regions of the M gene of TGEV, the N gene of PEDV, and the 3D gene of PKV were selected to design specific primers and probes. After optimizing the reaction conditions, the method's specificity, sensitivity, and reproducibility were evaluated. The triplex RT-qPCR method did not show a significant difference in PCR efficiency compared to the single RT-qPCR method. The method is specific to TGEV, PKV, and PEDV, exhibits no cross-reactivity with other pathogens, and demonstrates satisfactory sensitivity and reproducibility; the limit of detection (LOD) of PEDV, TGEV, and PKV is 11.42 copies/μL. Furthermore, the performance of the triplex RT-qPCR assay was compared with the Chinese standard single-assay method for detecting TGEV, PKV, and PEDV, showing complete consistency between the two methods (100% compliant). Subsequently, 1502 clinical diarrhea samples were collected from the Guangxi Zhuang Autonomous Region to investigate the local prevalence of TGEV, PKV, and PEDV and the positive rates were 16.38% (246/1502), 1.46% (22/1502), and 45.14% (678/1502), respectively. Co-infection of PEDV and PKV were most common, with a rate of 12.12% (182/1502). This study presents a valuable method for the rapid and simultaneous identification of PEDV, TGEV, and PKV in clinical animal farming practices, and provides a reassessment of the epidemiology of these diarrhea-causing viral pathogens in the Guangxi Zhuang Autonomous Region.</p>\",\"PeriodicalId\":23694,\"journal\":{\"name\":\"Veterinary Sciences\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2024-09-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11435592/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Veterinary Sciences\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.3390/vetsci11090413\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"VETERINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Veterinary Sciences","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.3390/vetsci11090413","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
Establishment and Application of a Triplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of PEDV, TGEV and PKV.
The pathogens responsible for porcine viral diarrhea are diverse, causing significant economic losses to the pig industry. PEDV and TGEV are well-known pathogens causing diarrheal diseases in pigs, leading to significant economic losses in the breeding industry. In contrast, the newly identified diarrhea virus, PKV, has not garnered as much attention. However, co-infection of PKV with PEDV results in more severe symptoms in piglets, such as acute gastroenteritis, and promotes increased replication of PEDV. Rapid and accurate diagnosis of viral diarrhea is essential for farms to identify pathogens early and mitigate economic losses. This study describes the development of a triplex real-time fluorescent quantitative RT-qPCR technique that can simultaneously detect three RNA viruses associated with porcine viral diarrhea: PEDV, TGEV, and PKV. To establish the triplex RT-qPCR method for the simultaneous detection and identification of the above three diarrhea viruses, conserved regions of the M gene of TGEV, the N gene of PEDV, and the 3D gene of PKV were selected to design specific primers and probes. After optimizing the reaction conditions, the method's specificity, sensitivity, and reproducibility were evaluated. The triplex RT-qPCR method did not show a significant difference in PCR efficiency compared to the single RT-qPCR method. The method is specific to TGEV, PKV, and PEDV, exhibits no cross-reactivity with other pathogens, and demonstrates satisfactory sensitivity and reproducibility; the limit of detection (LOD) of PEDV, TGEV, and PKV is 11.42 copies/μL. Furthermore, the performance of the triplex RT-qPCR assay was compared with the Chinese standard single-assay method for detecting TGEV, PKV, and PEDV, showing complete consistency between the two methods (100% compliant). Subsequently, 1502 clinical diarrhea samples were collected from the Guangxi Zhuang Autonomous Region to investigate the local prevalence of TGEV, PKV, and PEDV and the positive rates were 16.38% (246/1502), 1.46% (22/1502), and 45.14% (678/1502), respectively. Co-infection of PEDV and PKV were most common, with a rate of 12.12% (182/1502). This study presents a valuable method for the rapid and simultaneous identification of PEDV, TGEV, and PKV in clinical animal farming practices, and provides a reassessment of the epidemiology of these diarrhea-causing viral pathogens in the Guangxi Zhuang Autonomous Region.
期刊介绍:
Veterinary Sciences is an international and interdisciplinary scholarly open access journal. It publishes original that are relevant to any field of veterinary sciences, including prevention, diagnosis and treatment of disease, disorder and injury in animals. This journal covers almost all topics related to animal health and veterinary medicine. Research fields of interest include but are not limited to: anaesthesiology anatomy bacteriology biochemistry cardiology dentistry dermatology embryology endocrinology epidemiology genetics histology immunology microbiology molecular biology mycology neurobiology oncology ophthalmology parasitology pathology pharmacology physiology radiology surgery theriogenology toxicology virology.