标准化并评估了两个基于SYBRGreen的多重实时PCR检测方法,以检测来自四个犬类血液寄生虫(Babesiagibsoni,Vogeli巴贝西亚,犬埃里希氏菌和肝虫犬),以及旁遮普邦某些地区的狗的内部控制,印度。126bp的扩增子,337bp,234bp和106bp对应于双歧杆菌(18SrRNA基因),B.vogeli(18SrRNA基因),E.canis(virB9基因),并获得犬H.canis(18SrRNA基因),没有任何非特异性扩增。从狗的200个血液样本的显微镜评估显示了B.gibsoni的患病率,E.canis和H.canis为1.5%,1.5%和1.0%,分别,而在多重实时PCR检测中,B.Vogeli,E.canis,和H.canis为8.0%,1.5%,3.5%和23.5%,分别,同时感染B.gibsoni和H.canis(3.5%);E.canis和H.canis(2.0%)和B.gibsoni,B.Vogeli,E.canis,和H.canis(0.5%)。多重实时PCR检测对显微镜的诊断灵敏度。犬E.canis和犬H.canis为100%,而对B.gibsoni的特异性,B.Vogeli,E.canis,H.canis是93%,100%,98%和77%,分别,揭示了各自的协议强度是“公平的”,“轻微”,根据卡伯值统计,“中等”和“轻微”,数据具有统计学意义,用于检测Gibsoni芽孢杆菌和E.canis感染,根据费希尔的精确检验。多重PCR检测DNA的灵敏度分别为8.59×105和9.9×106个拷贝,分别,以及B.gibsoni和H.canis的1.15×106和3.41×105份,分别。风险因素评估。年龄,性别,品种,季节和位置与这些血寄生虫的患病率没有显着关联,除了B.vogeli,E.canis和H.canis在位置上发现了重要的关联,年龄和品种,分别通过多重实时PCR检测。
Two multiplex SYBR Green based real-time PCR assays were standardized and evaluated to detect DNA from four canine haemoparasites (Babesia gibsoni, Babesia vogeli, Ehrlichia canis and Hepatozoon canis), along with internal controls from dogs from selected districts of Punjab state, India. Amplicons of 126 bp, 337 bp, 234 bp and 106 bp corresponding to B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), E. canis (virB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. Microscopic evaluation of 200 blood samples from dogs revealed the prevalence of B. gibsoni, E. canis and H. canis as 1.5%, 1.5% and 1.0%, respectively, while with the multiplex real-time PCR assays the values for B. gibsoni, B. vogeli, E. canis, and H. canis were 8.0%, 1.5%, 3.5% and 23.5%, respectively, with concurrent infections of B. gibsoni and H. canis (3.5%); E. canis and H. canis (2.0%) and B. gibsoni, B. vogeli, E. canis, and H. canis (0.5%). The diagnostic sensitivity of the multiplex real-time PCR assays with respect to microscopy in the detection of B. gibsoni, E. canis and H. canis was 100% while the specificity for B. gibsoni, B. vogeli, E. canis, and H. canis was 93%, 100%, 98% and 77%, respectively, revealing the respective strength of agreement as ″fair″, ″slight″, ″moderate″ and ″slight″ by kappa value statistics, and the data were statistically significant, for detection of B. gibsoni and E. canis infections, by Fisher\'s exact test. The analytical sensitivity of the multiplex PCR assays in detection of DNAs was 8.59 × 105 and 9.9 × 106 copies for B. vogeli and E. canis, respectively, and 1.15 × 106 and 3.41 × 105 copies for B. gibsoni and H. canis, respectively. Assessment of risk factors viz. age, sex, breed, season and locations showed no significant association with the prevalence of these haemoparasites except for B. vogeli, E. canis and H. canis where significant associations were found for location, age and breed, respectively by multiplex real-time PCR assays.