Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 141004, Punjab, India.
Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 141004, Punjab, India.
Acta Trop. 2020 Dec;212:105713. doi: 10.1016/j.actatropica.2020.105713. Epub 2020 Sep 16.
A multiplex PCR assay was standardized and evaluated to simultaneously detect the DNA of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs of selected districts of Punjab state, India. Amplicons of 602 bp, 380 bp and 306 bp corresponding to B. vogeli (18S rRNA gene), E. canis (VirB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. The results of multiplex PCR assay were further compared with the corresponding singleplex PCR assay. The diagnostic sensitivity and specificity of multiplex PCR assay with respect to singleplex PCR assay in the detection of B. vogeli, E. canis and H. canis varied from 50% to 100% and 92.08% to 98.79%, respectively revealing "moderate" to "very good" agreement by kappa value statistics. Blood samples from 322 dogs collected from selected districts of Punjab state, India, when screened by microscopy revealed the prevalence of B. vogeli, E. canis and H. canis as 0.31%, 0.93% and 1.86%, respectively whereas with multiplex PCR assay the values were 0.93%, 10.24% and 4.65%, respectively, with concurrent infection of E. canis & H. canis (1.86%) and B. vogeli & E. canis (0.31%). The diagnostic sensitivity and specificity of multiplex PCR assay with respect to microscopy in the detection of B. vogeli, E. canis and H. canis varied from 69.15% to 100% and 85.11% to 92.33%, respectively revealing "fair" agreement by kappa value statistics and the data was statistically significant. The analytical sensitivity of multiplex PCR assay in the detection of B. vogeli, E. canis and H. canis was 100 pg, 10 pg and 0.1 pg, respectively, whereas the values for the singleplex counterpart were 0.1 pg, 0.01 pg and 0.01 pg. Furthermore, various risk factors viz. age, breed, sex, season and districts were non-significantly associated with the prevalence of these haemoparasites except for E. canis that revealed a significant association with districts by multiplex PCR assay. Therefore the multiplex PCR assay developed may be useful in identification of the aetiological agents of these diseases during their early phase, which may in turn be useful in development of better health care and appropriate treatment of suspected dogs, particularly in endemic regions.
我们建立并评估了一种多重 PCR 检测方法，用于同时检测来自印度旁遮普邦选定地区犬的贝氏巴贝斯虫、犬埃立克体和犬无形体的 DNA。获得了 602 bp、380 bp 和 306 bp 的扩增子，分别对应于贝氏巴贝斯虫（18S rRNA 基因）、犬埃立克体（VirB9 基因）和犬无形体（18S rRNA 基因），没有任何非特异性扩增。多重 PCR 检测方法的结果与相应的单重 PCR 检测方法进行了比较。多重 PCR 检测方法在检测贝氏巴贝斯虫、犬埃立克体和犬无形体时的诊断灵敏度和特异性在 50%至 100%之间，kappa 值统计显示与单重 PCR 检测方法的一致性为“中度”至“非常好”。从印度旁遮普邦选定地区采集的 322 份犬血样经显微镜检查发现，贝氏巴贝斯虫、犬埃立克体和犬无形体的流行率分别为 0.31%、0.93%和 1.86%，而用多重 PCR 检测方法的流行率分别为 0.93%、10.24%和 4.65%，同时存在犬埃立克体和犬无形体（1.86%）以及贝氏巴贝斯虫和犬埃立克体（0.31%）的混合感染。多重 PCR 检测方法在检测贝氏巴贝斯虫、犬埃立克体和犬无形体时的诊断灵敏度和特异性在 69.15%至 100%和 85.11%至 92.33%之间，kappa 值统计显示一致性为“中等”，数据具有统计学意义。多重 PCR 检测方法在检测贝氏巴贝斯虫、犬埃立克体和犬无形体时的分析灵敏度分别为 100 pg、10 pg 和 0.1 pg，而单重 PCR 检测方法的灵敏度分别为 0.1 pg、0.01 pg 和 0.01 pg。此外，年龄、品种、性别、季节和地区等各种风险因素与这些血液寄生虫的流行率均无显著相关性，除了犬埃立克体，其与多重 PCR 检测方法的地区显著相关。因此，该方法可能有助于在疾病的早期阶段识别这些疾病的病因，这反过来又有助于在流行地区制定更好的保健和适当的治疗疑似犬的措施。
