Liting Ke , Guangzheng Hou , Pengcheng Cao , Yanlei Ding , Changxu Zhao , Fei Wang , Yingqiu Liu , Yunpeng Fan , Qiqi Liu
{"title":"建立并验证针对 11 种绵羊和山羊病原体的实时荧光 PCR 冻干型检测方法。","authors":"Liting Ke , Guangzheng Hou , Pengcheng Cao , Yanlei Ding , Changxu Zhao , Fei Wang , Yingqiu Liu , Yunpeng Fan , Qiqi Liu","doi":"10.1016/j.tvjl.2024.106255","DOIUrl":null,"url":null,"abstract":"<div><div>Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99–1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for <em>M. ovis</em>, Pathogenic <em>Leptospira</em>, <em>C. burnetii</em>, BTV, PPRV, <em>Brucella</em>, exJSRV, <em>C. abortus</em>, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00 %; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were <em>Mycoplasma ovis</em> (<em>M. ovis</em>), Pathogenic <em>Leptospira</em>, <em>Coxiella burnetii</em> (<em>C. burnetii</em>), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), <em>Brucella</em>, exogenous jaagsiekte sheep retrovirus (exJSRV), <em>Chlamydia abortus</em> (<em>C. abortus</em>), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).</div></div>","PeriodicalId":23505,"journal":{"name":"Veterinary journal","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens\",\"authors\":\"Liting Ke , Guangzheng Hou , Pengcheng Cao , Yanlei Ding , Changxu Zhao , Fei Wang , Yingqiu Liu , Yunpeng Fan , Qiqi Liu\",\"doi\":\"10.1016/j.tvjl.2024.106255\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99–1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for <em>M. ovis</em>, Pathogenic <em>Leptospira</em>, <em>C. burnetii</em>, BTV, PPRV, <em>Brucella</em>, exJSRV, <em>C. abortus</em>, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00 %; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were <em>Mycoplasma ovis</em> (<em>M. ovis</em>), Pathogenic <em>Leptospira</em>, <em>Coxiella burnetii</em> (<em>C. burnetii</em>), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), <em>Brucella</em>, exogenous jaagsiekte sheep retrovirus (exJSRV), <em>Chlamydia abortus</em> (<em>C. abortus</em>), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).</div></div>\",\"PeriodicalId\":23505,\"journal\":{\"name\":\"Veterinary journal\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2024-10-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Veterinary journal\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1090023324001941\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"VETERINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Veterinary journal","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1090023324001941","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
Establishment and validation of a real-time fluorescent PCR freeze-dried type assay for 11 sheep and goats pathogens
Small ruminants are economic mainstay in developing countries because of its direct contribution to food security, but the occurrence of epidemics poses a continuous threat. Early diagnosis can strengthen the prevention of zoonosis. Therefore, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens in this study. The results showed that there was no non-specific amplification in systems containing different pathogen positive controls; The areas under the ROC curves were all in the range of 0.99–1.00; The LOD counted by Digital PCR were 194, 96, 84, 40, 265, 68, 208, 236, 118, 53 and 723 copies/mL for M. ovis, Pathogenic Leptospira, C. burnetii, BTV, PPRV, Brucella, exJSRV, C. abortus, ORFV, FMDV and CPV, respectively. The coefficients of variation of both intra-group and inter-group replicate tests were less than 5.00 %; The accelerated thermostatic lyophilization test was used to predict the validity period; The LOD of each positive pathogen was still detected after 6 days at 56 ℃, suggesting that the validity period can be at least 567 days at room temperature. In this study, a high-sensitivity real-time fluorescent PCR freeze-dried type assay that can be stored and transported at room temperature was developed for 11 sheep and goats pathogens, which were Mycoplasma ovis (M. ovis), Pathogenic Leptospira, Coxiella burnetii (C. burnetii), Bluetongue virus (BTV), Peste des pestis ruminants virus (PPRV), Brucella, exogenous jaagsiekte sheep retrovirus (exJSRV), Chlamydia abortus (C. abortus), Orf virus (ORFV), Foot-and-mouth disease virus (FMDV), and Capripox virus (CPV).
期刊介绍:
The Veterinary Journal (established 1875) publishes worldwide contributions on all aspects of veterinary science and its related subjects. It provides regular book reviews and a short communications section. The journal regularly commissions topical reviews and commentaries on features of major importance. Research areas include infectious diseases, applied biochemistry, parasitology, endocrinology, microbiology, immunology, pathology, pharmacology, physiology, molecular biology, immunogenetics, surgery, ophthalmology, dermatology and oncology.