Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus using a multiplex PCR assay

Liu, Cong-Nuan, Lou, Zhong-Zi, Li, Li, Yan, Hong-Bin, Blair, David, Lei, Meng-Tong, Cai, Jin-Zhong, Fan, Yan-Lei, Li, Jian-Qiu, Fu, Bao-Quan, Yang, Yu-Rong, McManus, Donald P., and Jia, Wan-Zhong (2015) Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus using a multiplex PCR assay. PLoS Neglected Tropical Diseases, 9 (9). pp. 1-14.

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Abstract

Background: 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.

Methodology/Principal Findings: 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.

Conclusions/Significance: 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.

Item ID: 41820
Item Type: Article (Refereed Research - C1)
Additional Information:

© 2015 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ISSN: 1935-2735
Funders: Gansu Provincial Key Science and Technology Projects (GPKSTP), Special Fund for Agro-Scientific Research in the Public Interest (SFASR), Science Fund for Creative Research Groups of Gansu Province (SFCRGG), Natural Science Foundation of China (NSFC), National Health and Medical Research Council of Australia (NHMRC)
Projects and Grants: GPKSTP Grant No. 120NKDA039, SFASR Grant No. 201303037, SFASR Grant No. 200903036-07, SFCRGG Grant No. 120RJIA006, NSFC Grant No. 30960339, NHMRC Grant No. APP-1009539
Date Deposited: 08 Dec 2015 18:41
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110803 Medical Parasitology @ 100%
SEO Codes: 92 HEALTH > 9204 Public Health (excl. Specific Population Health) > 920404 Disease Distribution and Transmission (incl. Surveillance and Response) @ 100%
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