Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis
Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis
复制标题
DOI：
10.1038/s41598-020-60997-1
复制
发表时间：
2020-03-05
影响因子：
4.6
通讯作者：
Jiang, Ning
中科院分区：
文献类型：
--
作者：
Lei, Rong;Wang, Xinyi;Jiang, Ning
马梨形虫病（EP）是由马泰勒虫（T. equi）和卡氏巴氏巴氏菌（Babelus caballi，B. caballi）。传染性带菌者并不总是有症状的，这意味着非流行区存在风险。EP的监管检测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属的交叉反应性，这些方法缺乏特异性。在这项研究中，我们提出了一种实时定量重组酶聚合酶扩增（qRPA）方法快速同时检测两个T。equi和B.卡巴利。该方法利用特异性引物和探针，分别对两种T. equi和B. caballi、T. equi和B. caballi的设计和评价。使用含有靶基因的pUC 57质粒DNA评价qRPA的灵敏度。对于pUC 57-bc 48基因DNA，浓度范围为0.2 ng（4.1 x 10（7）DNA拷贝）至2.0 fg（4.1 x 10（1）DNA拷贝）时，R-2值为0.983。对于pUC 57-ema基因DNA，浓度范围为0.2 ng（5.26 x 10（7）DNA拷贝）至2.0 fg（5.26 x 10（2）DNA拷贝）的R-2值为0.993。对于pUC 57-Bc 18 S基因DNA，浓度范围为2.0 ng（4.21 x 10（8）DNA拷贝）至2.0 fg（4.21 x 10（2）DNA拷贝）的R-2值为0.976。对于pUC 57-Te 18 S基因DNA，浓度范围为2.0 ng（4.16 x 10（8）DNA拷贝）至2.0 fg（4.16 x 10（2）DNA拷贝）时，R-2值为0.952（图S3 b）。结果表明，针对B的bc 48基因的引物和探针均能扩增出正确的引物和探针。caballi和T. equi是在一个反应中进行双重qRPA分析的最佳组合。建立的双链qRPA检测方法特异性好，对几种类似寄生虫均为阴性扩增。对于从真实的马血标本中提取的DNA，该qRPA方法具有与传统qPCR相当的灵敏度，但获得阳性扩增的操作过程更简单、更快速。qRPA，包括这里描述的双链策略，可以快速识别引起EP的T。equi和B. caballi，显示出巨大的潜力，现场EP筛选的马匹。
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-2 value was 0.983 for the concentration range 0.2 ng (4.1 x 10(7) DNA copies) to 2.0 fg (4.1 x 10(1) DNA copies). For the pUC57-ema gene DNA, the R-2 value was 0.993 for the concentration range 0.2 ng (5.26 x 10(7) DNA copies) to 2.0 fg (5.26 x 10(2) DNA copies). For the pUC57-Bc18S gene DNA the R-2 value was 0.976 for the concentration range 2.0 ng (4.21 x 10(8) DNA copies) to 2.0 fg (4.21 x 10(2) DNA copies). For the pUC57-Te18S gene DNA, the R-2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 x 10(8) DNA copies) to 2.0 fg (4.16 x 10(2) 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.