马螺旋体病(EP)是由三种尖丛原生动物寄生虫引起的蜱传播疾病,Theileriaequi(T.equi),巴贝西亚(B.caballi)和T.haneyi,会引起类似的临床症状.有五种已知的T.equi组(包括T.haneyi)和三种B.caballi的18SrRNA基因型。已经开发了基于18SrRNA分析的检测EP的实时PCR方法,但是这些方法不能在中国检测到所有基因型的EP,特别是T.equi的基因型A。在这项研究中,开发了一种双重实时PCR检测方法,用于同时检测和区分T.equi和B.caballi。基于包括中国菌株在内的所有基因型的马氏T.equi和B.caballi的保守18SrRNA基因序列,设计了用于该双重实时PCR检测的引物和探针。在这种方法中使用了双淬火探针,其提供更少的背景和更多的信号以减少相对于单一淬灭探针的假阳性的数量。新开发的实时PCR检测方法具有良好的特异性,灵敏度,可重复性和再现性。通过与巢式PCR测定和先前开发的EP和测序结果的实时PCR比较,在分析2019年至2020年在中国11个省和地区收集的506个临床样本时,进一步验证了实时PCR测定。根据临床表现,双重实时PCR测定与nPCR测定或先前开发的实时PCR测定之间的一致性为92.5%(T。equi)和99.4%(B.caballi)或87.4%(T.equi)和97.2%(B.卡瓦利)。检测结果显示,马氏T.equi的阳性率为43.87%(222/506)(10基因型A,1基因型B,4基因型C,207基因型E),而B.caballi为5.10%(26/506)(26基因型A),T.equi和B.caballi共感染率为2.40%(12/506)。所建立的方法有助于准确诊断,马中T.equi和B.caballi感染的病原监测和流行病学调查。
Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.