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      A multiplex PCR assay for the simultaneous detection of Taenia hydatigena, T. multiceps, T. pisiformis, and Dipylidium caninum infections

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          Abstract

          Background

          Taenia hydatigena, T. multiceps, T. pisiformis, and Dipylidium caninum are four common large and medium-sized tapeworms parasitizing the small intestine of dogs and other canids. These parasites cause serious impact on the health and development of livestock. However, there are, so far, no commercially available molecular diagnostic kits capable of simultaneously detecting all four parasites in dogs. The aim of the study was therefore to develop a multiplex PCR assay that will accurately detect all four cestode infections in one reaction.

          Methods

          Specific primers for a multiplex PCR were designed based on corresponding mitochondrial genome sequences, and its detection limit was assessed by serial dilutions of the genomic DNAs of tapeworms examined. Furthermore, field samples of dog feces were tested using the developed assay.

          Results

          A multiplex polymerase chain reaction (PCR) assay was developed based on mitochondrial DNA ( mtDNA) that accurately and simultaneously identify four cestode species in one reaction using specific fragment sizes of 592, 385, 283, and 190 bp for T. hydatigena, T. multiceps, T. pisiformis, and D. caninum, respectively. The lowest DNA concentration detected was 1 ng for T. hydatigena, T. multiceps and T. pisiformis, and 0.1 ng for D. caninum in a 25 μl reaction system. This assay offers high potential for the rapid detection of these four tapeworms in host feces simultaneously.

          Conclusions

          This study provides an efficient tool for the simultaneous detection of T. hydatigena, T. multiceps, T. pisiformis, and D. caninum. The assay will be potentially useful in epidemiological studies, diagnosis, and treatment of these four cestodes infections during prevention and control program.

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          Most cited references 36

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          Multiplex PCR: critical parameters and step-by-step protocol.

          By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.
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            Ecology and Life Cycle Patterns of Echinococcus Species.

            The genus Echinococcus is composed of eight generally recognized species and one genotypic cluster (Echinococcus canadensis cluster) that may in future be resolved into one to three species. For each species, we review existing information on transmission routes and life cycles in different geographical contexts and - where available - include basic biological information of parasites and hosts (e.g., susceptibility of host species). While some Echinococcus spp. are transmitted in life cycles that involve predominantly domestic animals (e.g., dog - livestock cycles), others are wildlife parasites that do or do not interact with domestic transmission. In many cases, life cycle patterns of the same parasite species differ according to geography. Simple life cycles contrast with transmission patterns that are highly complex, involving multihost systems that may include both domestic and wild mammals. Wildlife transmission may be primary or secondary, i.e., resulting from spillovers from domestic animals. For most of the species and regions, existing information does not yet permit a conclusive description of transmission systems. Such data, however, would be highly relevant, e.g., for anticipation of geographical changes of the presence and frequency of these parasites in a warming world, or for initiating evidence-based control strategies.
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              Mitogenomics: digging deeper with complete mitochondrial genomes.

              Mitochondrial genomes are being used to study increasingly ancient divergences among animal groups. Recent studies of complete mitochondrial DNA sequences have arrived at somewhat heretical conclusions, raising questions about the use of mitochondrial gene sequences for studying the relationships among highly divergent lineages. Other studies have documented convergent evolution of mitochondrial gene order, casting doubt on the use of these characters for phylogenetic analysis. The use of mitochondrial genomes for studying such deep divergences is coming under increased scrutiny, and these novel results need to be confirmed with data from nuclear genes.
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                Author and article information

                Contributors
                zhuguoqiang915@163.com
                lili03@caas.cn
                asekhaenj@gmail.com
                643058923@qq.com
                liwenhui@caas.cn
                zhangnianzhang@caas.cn
                fubaoquan@caas.cn
                yanhongbin@caas.cn
                +86-931-8312212 , jiawanzhong@caas.cn
                Journal
                BMC Infect Dis
                BMC Infect. Dis
                BMC Infectious Diseases
                BioMed Central (London )
                1471-2334
                16 October 2019
                16 October 2019
                2019
                : 19
                Affiliations
                [1 ]ISNI 0000 0001 0526 1937, GRID grid.410727.7, State Key Laboratory of Veterinary Etiological Biology/ Key Laboratory of Veterinary Parasitology of Gansu Province/ Lanzhou Veterinary Research Institute, , CAAS, ; Lanzhou, 730046 Gansu Province People’s Republic of China
                [2 ]Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease, Yangzhou, 225009 Jiangsu Province, People’s Republic of China
                Article
                4512
                10.1186/s12879-019-4512-3
                6796438
                31619188
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: National Key Research and Development Program of China
                Award ID: 2017YFD0501301
                Award Recipient :
                Funded by: National Key Basic Research Program (973 Program) of China
                Award ID: 2015CB150300
                Award Recipient :
                Funded by: Gansu Provincial Key Science and Technology Projects
                Award ID: 1203NKDA039
                Award Recipient :
                Funded by: Science Fund for Creative Research Groups of Gansu Province
                Award ID: 1210RJIA006
                Award Recipient :
                Funded by: Central Public-interest Scientific Institution Basal Research Fund
                Award ID: 1610312017001
                Award Recipient :
                Funded by: Central Public-interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences (CN)
                Award ID: 1610312016012
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2019

                Infectious disease & Microbiology

                multiplex pcr, canids, tapeworms, mitochondrial dna

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