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。

IF 2.3 2区 农林科学 Q1 VETERINARY SCIENCES Veterinary journal Pub Date : 2024-10-17 DOI:10.1016/j.tvjl.2024.106255
Liting Ke , Guangzheng Hou , Pengcheng Cao , Yanlei Ding , Changxu Zhao , Fei Wang , Yingqiu Liu , Yunpeng Fan , Qiqi Liu
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引用次数: 0

摘要

小反刍兽疫是发展中国家的经济支柱,因为它直接关系到粮食安全,但疫情的发生也构成了持续的威胁。早期诊断可以加强人畜共患病的预防。因此,本研究针对 11 种绵羊和山羊病原体开发了一种可在室温下储存和运输的高灵敏度实时荧光 PCR 冻干型检测方法。结果表明,在含有不同病原体阳性对照的系统中不存在非特异性扩增;ROC 曲线下的面积均在 0.99 至 1.00;数字 PCR 对猫鼬病毒、钩端螺旋体病原体、烧伤蜱、BTV、PPRV、布鲁氏菌、exJSRV、流产蜱、ORFV、FMDV 和 CPV 的 LOD 分别为 194、96、84、40、265、68、208、236、118、53 和 723 拷贝/毫升。组内和组间重复试验的变异系数均小于 5.00%;采用加速恒温冻干试验预测有效期;在 56 ℃ 下 6 天后仍能检测到各阳性病原体的 LOD,表明在室温下有效期至少可达 567 天。本研究针对 11 种绵羊和山羊病原体开发了一种可在室温下储存和运输的高灵敏度实时荧光 PCR 冻干型检测方法,这 11 种病原体分别是绵羊支原体(M. ovis)、致病性钩端螺旋体(Pathogenic Leptospira)、烧伤科克西氏菌(C.burnetii)、蓝舌病病毒(BTV)、反刍动物鼠疫病毒(PPRV)、布鲁氏菌、外源绵羊逆转录病毒(exJSRV)、流产衣原体(C. abortus)、Orf 病毒(ORFV)、口蹄疫病毒(FMDV)和痘病毒(CPV)。
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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).
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来源期刊
Veterinary journal
Veterinary journal 农林科学-兽医学
CiteScore
4.10
自引率
4.50%
发文量
79
审稿时长
40 days
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.
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