Shi Xiju, Liu Xuming, Wang Qin, Das Amaresh, Ma Guiping, Xu Lu, Sun Qing, Peddireddi Lalitha, Jia Wei, Liu Yanhua, Anderson Gary, Bai Jianfa, Shi Jishu
Beijing Entry-Exit Inspection & Quarantine Bureau, Beijing, China; Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States.
J Virol Methods. 2016 Oct;236:258-265. doi: 10.1016/j.jviromet.2016.08.005. Epub 2016 Aug 6.
Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.
在养猪生产系统中，尤其是在集约化生产条件下，不同病原体的混合感染很常见。快速准确地检测和区分不同病原体对于流行病学监测、疾病管理以及进出口控制至关重要。在本研究中，我们开发并验证了一组多重实时PCR/RT-PCR检测方法，该方法由四个子检测板组成，每个子检测板可检测三种常见的猪病原体。该检测板可检测12种病毒或病毒血清型，即水疱性口炎病毒印第安纳株（VSV-IN）、水疱性口炎病毒新泽西株（VSV-NJ）、猪瘟病毒（SVDV）、猪瘟病毒（CSFV）、非洲猪瘟病毒（ASFV）、口蹄疫病毒（FMDV）、猪圆环病毒2型（PCV2）、猪细小病毒（PPV）、伪狂犬病病毒（PRV）、北美型猪繁殖与呼吸综合征病毒（PRRSV-NA）、欧洲型猪繁殖与呼吸综合征病毒（PRRSV-EU）和猪流感病毒（SIV）。所有单重和三重实时PCR反应的相关系数（R²）和PCR扩增效率均在可接受范围内。每个子检测板的单重和三重实时PCR检测方法之间的比较表明，多重检测对检测灵敏度没有显著干扰。对226份目标临床样本或4种病毒株以及91份非目标临床样本进行的特异性测试表明，该实时PCR检测板具有100%的特异性，未观察到交叉扩增。每个三重实时PCR的检测限对于DNA病毒而言每个反应小于10个拷贝，对于RNA病毒而言每个反应小于16个拷贝。新开发的多重实时PCR检测板还检测到了经其他检测方法确认的不同共感染组合。
