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首页> 外文期刊>Journal of Virological Methods >Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis
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Simultaneous detection of five pig viruses associated with enteric disease in pigs using EvaGreen real-time PCR combined with melting curve analysis

机译：使用EVAGREEN实时PCR结合熔化曲线分析，同时检测与猪中猪病患者相关的五种猪病毒

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摘要

In recent years, a series of porcine diarrhea viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), rotaviruses of group A (RVA), rotaviruses of group C (RVC), and porcine circovirus 2 (PCV2) caused enormous economic losses all over the world. While any of these viruses is capable to cause disease alone, there is often concurrent infection with more than one virus on pig farms. In this study, a multiplex real-time PCR method based on EvaGreen fluorescent dye and melting curve analysis was established to simultaneously detect these five viruses in a single closed tube. Five distinct melt peaks were obtained with different melting temperature (Tm) value corresponding to each of the five viruses. This method was highly sensitive to detect and distinguish TGEV, RVA, RVC, PEDV and PCV2 with the limits of detection ranging from 5 to 50 copies/mu L. The intra-assay and inter-assay reproducibility were good with coefficient of variation of Tm and cycle threshold values less than 0.32% and 2.86%, respectively. Testing of 90 field samples by the single and multiplex real-time PCR assays demonstrated a concordance of 91.1%. Thus, the EvaGreen multiplex real-time PCR is a rapid, sensitive and low-cost diagnostic tool for differential detection and routine surveillance of TGEV, RVA, RVC, PEDV and PCV2 in pigs.
机译：近年来，一系列猪腹泻病毒如猪流行性腹泻病毒（PEDV），传染性胃肠炎病毒（TGEV），A组（RVA）的旋转毒物，C组（RVC）的旋泻，和猪胃肠病毒2（PCV2）在世界各地造成了巨大的经济损失。虽然任何这些病毒​​能够单独引起疾病，但通常在养猪场上具有多种病毒的同时感染。在该研究中，建立了一种基于Evageen荧光染料和熔化曲线分析的多重实时PCR方法，同时检测单个封闭管中的这五种病毒。使用与五种病毒中的每种病毒相对应的不同熔化温度（TM）值获得五个不同的熔融峰。该方法对检测和区分TGEV，RVA，RVC，PEDV和PCV2具有高敏感性，检测范围范围为5至50拷贝/μL。测定内和测定间再现性具有TM的变异系数并且循环阈值分别小于0.32％和2.86％。通过单层和多重实时PCR测定检测90个田间样本证明了91.1％的一致性。因此，EVAGREEN多重实时PCR是TGEV，RVA，RVC，PEDV和PICV2的差异检测和常规监测的快速，灵敏和低成本的诊断工具。

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