Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay.
Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay.
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DOI：
10.1371/journal.pntd.0004084
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发表时间：
2015-09
影响因子：
3.8
通讯作者：
Jia WZ
中科院分区：
文献类型：
--
作者：
Liu CN;Lou ZZ;Li L;Yan HB;Blair D;Lei MT;Cai JZ;Fan YL;Li JQ;Fu BQ;Yang YR;McManus DP;Jia WZ
细粒棘球绦虫（Echinococcus granulosus stricto（s.s））、细粒棘球绦虫（E. multilocularis和E.在中国青藏高原上，志贺氏菌是常见的共同特有种，因此需要一种有效的工具来检测受感染的宿主并进行物种鉴定。建立了一种单管多重PCR检测方法，用于区分中间宿主和终末宿主中的棘球绦虫感染。E. granulosus、E. multilocularis和E.根据线粒体NADH脱氢酶亚基1（nad 1）、NADH脱氢酶亚基5（nad 5）和细胞色素c氧化酶亚基1（cox 1）基因序列设计了3个不同的表达载体。这种多重PCR准确地检测棘球蚴DNA，而不产生非特异性反应产物。PCR产物的预期大小为219（nad 1）、584（nad 5）和471（cox 1）bp。此外，多重PCR能够使用从犬宿主粪便样品中提取的原噬菌体DNA和粪DNA诊断多重感染。当使用从其他绦虫分离的DNA进行评价时，多重PCR的特异性为100%。测定了原虫卵和虫卵DNA的敏感性阈值，并计算为20 pg的DNA对E. granulosus和E. shiquicus，E.多室棘球蚴2枚;细粒棘球绦虫E.多房的实验性感染犬大肠埃希氏菌幼虫后17天和26天，粪蛋白DNA检测均为阳性。multilocularis和E. granulosus。本研究建立的多重PCR方法是鉴别大肠杆菌的有效工具。granulosus、E. multilocularis和E.和其他绦虫的区别。该方法可用于犬感染E.颗粒菌和E. multicularis使用粪便收集这些最终主机。它也可用于鉴定中间宿主中的棘球绦虫幼虫，这是一个通常无法通过目视检查确定物种的阶段。犬适应性肠道绦虫有细粒棘球绦虫、细粒棘球绦虫、细粒棘球绦虫。multilocularis和E.摘要水曲柳是我国西北地区特有植物。前两个物种可以导致人类致命的疾病。虽然E.迄今为止，没有报道称志贺氏菌会感染人类，但这三种菌都可以通过狗传播。狗与人之间的密切关系很容易导致人类感染。为了帮助包虫病的监测和管理，迫切需要有效的诊断方法。我们建立了一种单管多重PCR方法，用于准确鉴定和区分三种棘球绦虫，用于临床诊断和流行病学研究。
Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification. A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively. The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification of the Echinococcus metacestode larva in intermediate hosts, a stage that often cannot be identified to species on visual inspection. The canid adapted intestinal tapeworms, Echinococcus granulosus, E. multilocularis and E. shuiqucus are well known to be endemic in Northwestern China. The first two species can cause fatal disease in humans. Although E. shiquicus has not been reported to infect humans, all three species can be transmitted by dogs. The very close relationship between dogs and humans can readily lead to human infection. To aid the surveillance and management of echinococcosis, effective diagnostic approaches are urgently needed. We developed a single tube multiplex PCR assay for the accurate identification and discrimination of the three Echinococcus species for use in both clinical diagnosis and epidemiological studies.