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一种用于区分猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪圆环病毒3型以及……的四重实时逆转录聚合酶链反应检测方法的建立与应用

Establishment and Application of a Quadruplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, Porcine Circovirus Type 3, and .

作者信息

Wang Geng, Zhu Hechao, Zhan Cunlin, Chen Pin, Wu Bin, Peng Zhong, Qian Ping, Cheng Guofu

机构信息

College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.

Guangxi Yangxiang Co., Ltd., Guigang 537100, China.

出版信息

Microorganisms. 2024 Feb 20;12(3):427. doi: 10.3390/microorganisms12030427.

Abstract

Respiratory illnesses present a significant threat to porcine health, with co-infections involving Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), (), Porcine Circovirus Type 2 (PCV2), and Porcine Circovirus Type 3 (PCV3) acting as the primary causative agents. As a result, the precise diagnosis of PRRSV, PCV2, PCV3 and is of paramount importance in the prevention and control of respiratory diseases in swine. Therefore, we conducted a molecular bioinformatical analysis to concurrently detect and differentiate PRRSV, PCV2, PCV3 and . We selected the ORF6 gene of PRRSV, the ORF2 gene of PCV2 and PCV3, and the glutamate dehydrogenase (GDH) gene of as targets. Specific primers and probes were designed for each pathogen, and following meticulous optimization of reaction conditions, we established a multiple TaqMan fluorescence quantitative PCR detection method. Subsequently, we subjected this method to a comprehensive assessment, evaluating its specificity, sensitivity, and repeatability. The research results demonstrated that the established multiple TaqMan fluorescence quantitative PCR detection method displays displayed exemplary specificity, with no instances of cross-reactivity with other pathogens. The method's minimum detection concentrations for PRRSV, PCV2, PCV3, and were 2.80 × 10 copies/µL, 1.96 × 10 copies/µL, 2.30 × 10 copies/µL, and 1.75 × 10 copies/µL, respectively. When applied to the analysis of 30 clinical samples, the results closely mirrored those obtained through Chinese standard uniplex real-time qPCR detection method for PRRSV, as well as the general PCR methods for , PCV2, and PCV3. This study underscores the robust specificity, high sensitivity, and consistent stability of the multiple TaqMan fluorescence quantitative PCR detection method that we have developed. It is ideally suited to the clinical monitoring of PRRSV, PCV2, PCV3, and , and it carries significant importance in ongoing efforts to prevent and manage respiratory diseases in porcine populations.

摘要

呼吸道疾病对猪的健康构成重大威胁,猪繁殖与呼吸综合征病毒(PRRSV)、(此处原文缺失某种病原体名称)、猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)的合并感染是主要致病因素。因此,对PRRSV、PCV2、PCV3以及(此处原文缺失某种病原体名称)进行准确诊断对于猪呼吸道疾病的预防和控制至关重要。为此,我们进行了分子生物信息学分析,以同时检测和区分PRRSV、PCV2、PCV3以及(此处原文缺失某种病原体名称)。我们选择PRRSV的ORF6基因、PCV2和PCV3的ORF2基因以及(此处原文缺失某种病原体名称)的谷氨酸脱氢酶(GDH)基因作为靶点。针对每种病原体设计了特异性引物和探针,在对反应条件进行精心优化后,我们建立了一种多重TaqMan荧光定量PCR检测方法。随后,我们对该方法进行了全面评估,评估其特异性、敏感性和重复性。研究结果表明,所建立的多重TaqMan荧光定量PCR检测方法具有出色的特异性,与其他病原体无交叉反应情况。该方法对PRRSV、PCV2、PCV3以及(此处原文缺失某种病原体名称)的最低检测浓度分别为2.80×10拷贝/µL、1.96×10拷贝/µL、2.30×10拷贝/µL和1.75×10拷贝/µL。当应用于30份临床样本分析时,结果与通过中国标准的PRRSV单重实时定量PCR检测方法以及(此处原文缺失某种病原体名称)、PCV2和PCV3的常规PCR方法所获得的结果密切相符。本研究强调了我们所开发的多重TaqMan荧光定量PCR检测方法具有强大的特异性、高敏感性和一致的稳定性。它非常适合于PRRSV、PCV2、PCV3以及(此处原文缺失某种病原体名称)的临床监测,对于当前猪群呼吸道疾病的预防和管理工作具有重要意义。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d04/10971858/fe9e5ba07a26/microorganisms-12-00427-g001.jpg

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