Institute of Parasitic Diseases, Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, People's Republic of China.
Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
Infect Dis Poverty. 2019 Jul 30;8(1):68. doi: 10.1186/s40249-019-0580-2.
Echinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.
In the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.
The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/μl, 10 pg/μl and 5 pg/μl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.
The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.
由细粒棘球绦虫引起的包虫病是中国西北高地最重要的传染病之一。已知在该地区传播的与人类健康有关的物种仅为三种，即细粒棘球绦虫、多房棘球绦虫和加拿大棘球绦虫。为了实现包虫病的靶向治疗和控制，准确识别和区分这些物种非常重要。然而，目前现有的诊断方法在准确性或效率方面都没有达到理想的效果。
在本研究中，设计了一组针对中国三种人源性棘球蚴的引物。建立并评估了一步多重 PCR 检测方法的特异性和敏感性。共检测了 73 例临床和现场采集的人体寄生虫病变和 41 份粪便样本，以评估该方法的适用性。
多重 PCR 能够有效地从目标物种及其随机混合物中检测到个体 DNA，产物具有可区分的预期大小。当分别检测时，该方法对三种物种的检测限均为 5pg/μl。当使用相同浓度的目标物种 DNA 混合物作为模板时，可检测到的最低 DNA 量分别为 50pg/μl、10pg/μl 和 5pg/μl，用于 E. granulosus s. s.、E. multilocularis 和 E. canadensis。当使用作为对照模板的 8 种遗传上相近的物种的 DNA 时，没有观察到交叉反应。除感染 E. shiquicus 的样本外，所有样本的多重 PCR 鉴定结果均与原始测序结果一致，而 E. shiquicus 在开发的检测方法中显示阴性信号。在所检测的所有粪便样本中，有 16 份先前通过肉眼和显微镜检查发现为包虫阳性。在这 16 个样本中，有 13 个通过多重 PCR 得到确认，另外 3 个检测结果为阴性。此外，多重 PCR 还从其余 25 个无虫粪便样本中鉴定出 14 个阳性样本。
该方法具有快速诊断和大规模流行病学调查的优势，有望成为临床和监测系统中的一种有用工具。
