Chinese Academy of Inspection and Quarantine, Beijing, 100176, China.
Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, Key Laboratory of Zoonosis, Shenyang Agricultural University, Shenyang, 110866, China.
Sci Rep. 2020 Mar 5;10(1):4096. doi: 10.1038/s41598-020-60997-1.
Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R value was 0.983 for the concentration range 0.2 ng (4.1 × 10 DNA copies) to 2.0 fg (4.1 × 10 DNA copies). For the pUC57-ema gene DNA, the R value was 0.993 for the concentration range 0.2 ng (5.26 × 10 DNA copies) to 2.0 fg (5.26 × 10 DNA copies). For the pUC57-Bc18S gene DNA the R value was 0.976 for the concentration range 2.0 ng (4.21 × 10 DNA copies) to 2.0 fg (4.21 × 10 DNA copies). For the pUC57-Te18S gene DNA, the R value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 10 DNA copies) to 2.0 fg (4.16 × 10 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.
马梨形虫病（EP）是一种由蜱传播的原生动物引起的马的严重疾病，包括马泰勒虫（T. equi）和马巴贝斯虫（B. caballi）。感染携带者并非总是有症状，这意味着非地方性地区存在风险。EP 的监管测试包括马巴贝斯虫病的血清流行病学方法，但由于与其他巴贝斯虫种的交叉反应，这些方法缺乏特异性。在本研究中，我们提出了一种实时定量重组聚合酶扩增（qRPA）方法，用于快速同时检测 T. equi 和 B. caballi。在该方法中，设计并评估了针对 T. equi 和 B. caballi 的 18S rRNA 基因、T. equi 的 ema-1 基因和 B. caballi 的 bc48 基因的引物和探针。使用含有靶基因的 pUC57 质粒 DNA 评估 qRPA 的灵敏度。对于 pUC57-bc48 基因 DNA，R 值为 0.983，浓度范围为 0.2ng（4.1×10 DNA 拷贝）至 2.0fg（4.1×10 DNA 拷贝）。对于 pUC57-ema 基因 DNA，R 值为 0.993，浓度范围为 0.2ng（5.26×10 DNA 拷贝）至 2.0fg（5.26×10 DNA 拷贝）。对于 pUC57-Bc18S 基因 DNA，R 值为 0.976，浓度范围为 2.0ng（4.21×10 DNA 拷贝）至 2.0fg（4.21×10 DNA 拷贝）。对于 pUC57-Te18S 基因 DNA，R 值为 0.952（图 S3b），浓度范围为 2.0ng（4.16×10 DNA 拷贝）至 2.0fg（4.16×10 DNA 拷贝）。此外，还开发并优化了双 qRPA 分析，结果表明，针对 B. caballi 的 bc48 基因和 T. equi 的 18S rRNA 基因的引物和探针是在一个反应中进行双 qRPA 分析的最佳组合。所开发的双 qRPA 检测方法具有良好的特异性，对几种类似寄生虫的扩增呈阴性。对于从真实马血标本中提取的 DNA，该 qRPA 方法与传统 qPCR 相比具有相当的灵敏度，但操作过程更简单、更快，可获得阳性扩增。包括本文所述的双 qRPA 策略在内的 qRPA 可快速鉴定引起 EP 的 T. equi 和 B. caballi，显示出在现场筛查马 EP 的巨大潜力。
