Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis
Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs...
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| Veröffentlicht in: | Parasites & vectors 2017-08, Vol.10 (1), p.394-394, Article 394 |
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| creator | Mahittikorn, Aongart Thammasonthijarern, Nipa Roobthaisong, Amonrattana Udonsom, Ruenruetai Popruk, Supaluk Siri, Sukhontha Mori, Hirotake Sukthana, Yaowalark |
| description | Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum.
LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand.
Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697).
This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis. |
| doi_str_mv | 10.1186/s13071-017-2330-2 |
| format | Article |
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LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand.
Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697).
This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.</description><identifier>ISSN: 1756-3305</identifier><identifier>EISSN: 1756-3305</identifier><identifier>DOI: 10.1186/s13071-017-2330-2</identifier><identifier>PMID: 28835287</identifier><language>eng</language><publisher>England: BioMed Central Ltd</publisher><subject>Animals ; Coccidians ; Coccidiosis - diagnosis ; Coccidiosis - parasitology ; Coccidiosis - veterinary ; Colorimetry ; Coloring Agents - metabolism ; DNA Primers ; Dog Diseases - diagnosis ; Dog Diseases - parasitology ; Dogs ; Feces - parasitology ; Genes, Protozoan ; Genetic aspects ; Genetic testing ; Genotypes ; Host-parasite relationships ; Innovations ; Limit of Detection ; Molecular Diagnostic Techniques ; Neospora - genetics ; Neospora - virology ; Nucleic Acid Amplification Techniques - methods ; Real-Time Polymerase Chain Reaction - methods ; Sensitivity and Specificity ; Temperature ; Thailand</subject><ispartof>Parasites & vectors, 2017-08, Vol.10 (1), p.394-394, Article 394</ispartof><rights>COPYRIGHT 2017 BioMed Central Ltd.</rights><rights>The Author(s). 2017</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c500t-b963b9505398f8832c4616df7303b5dd4030c832a04b3677d4e2ad6e6bb8625f3</citedby><cites>FETCH-LOGICAL-c500t-b963b9505398f8832c4616df7303b5dd4030c832a04b3677d4e2ad6e6bb8625f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569544/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569544/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27923,27924,53790,53792</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28835287$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Mahittikorn, Aongart</creatorcontrib><creatorcontrib>Thammasonthijarern, Nipa</creatorcontrib><creatorcontrib>Roobthaisong, Amonrattana</creatorcontrib><creatorcontrib>Udonsom, Ruenruetai</creatorcontrib><creatorcontrib>Popruk, Supaluk</creatorcontrib><creatorcontrib>Siri, Sukhontha</creatorcontrib><creatorcontrib>Mori, Hirotake</creatorcontrib><creatorcontrib>Sukthana, Yaowalark</creatorcontrib><title>Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis</title><title>Parasites & vectors</title><addtitle>Parasit Vectors</addtitle><description>Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum.
LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand.
Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697).
This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.</description><subject>Animals</subject><subject>Coccidians</subject><subject>Coccidiosis - diagnosis</subject><subject>Coccidiosis - parasitology</subject><subject>Coccidiosis - veterinary</subject><subject>Colorimetry</subject><subject>Coloring Agents - metabolism</subject><subject>DNA Primers</subject><subject>Dog Diseases - diagnosis</subject><subject>Dog Diseases - parasitology</subject><subject>Dogs</subject><subject>Feces - parasitology</subject><subject>Genes, Protozoan</subject><subject>Genetic aspects</subject><subject>Genetic testing</subject><subject>Genotypes</subject><subject>Host-parasite relationships</subject><subject>Innovations</subject><subject>Limit of Detection</subject><subject>Molecular Diagnostic Techniques</subject><subject>Neospora - genetics</subject><subject>Neospora - virology</subject><subject>Nucleic Acid Amplification Techniques - methods</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Sensitivity and Specificity</subject><subject>Temperature</subject><subject>Thailand</subject><issn>1756-3305</issn><issn>1756-3305</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkl2L1DAUhoso7jr6A7yRgDfrRdd8NGl7Iyzj18KCsup1SNPTmUibdJN01B_k__SMsy47IIF8Pucl5-UtiueMnjPWqNeJCVqzkrK65ELQkj8oTlktVYkH-fDe_qR4ktJ3ShVtpXpcnPCmEZI39Wnx-y3sYAzzBD6TMBBDxhDmcoLemQw9cSnkLcTJjMRM8-gGZ012wZMMduvdzQLE-J7YMM0mIuzJD5e35GYxPruM6A5IBDOW2U1APq-vyRAiQUkSzex6snNpQe0eUO-vLv7BGu88EA8hzSGG5NLT4tFgxgTPbtdV8e39u6_rj-XVpw-X64ur0kpKc9m1SnStpFK0zYAtclsppvqhFlR0su8rKqjFa0OrTqi67ivgpleguq5RXA5iVbw56M5LhxZYNCWaUc_RTSb-0sE4ffzi3VZvwk5LqVpZVShwdisQA3qTsp5csjCOBrtZkmat4Ew1rVCIvjygGzOCdn4IqGj3uL6QjPGac9Ygdf4fCkcPk7PBw-Dw_qjg1VEBMhl-5o1ZUtKXX66PWXZgLbqcIgx3nTKq9wnTh4RpTJjeJwynVfHivkV3Ff8iJf4Ad0DOYw</recordid><startdate>20170823</startdate><enddate>20170823</enddate><creator>Mahittikorn, Aongart</creator><creator>Thammasonthijarern, Nipa</creator><creator>Roobthaisong, Amonrattana</creator><creator>Udonsom, Ruenruetai</creator><creator>Popruk, Supaluk</creator><creator>Siri, Sukhontha</creator><creator>Mori, Hirotake</creator><creator>Sukthana, Yaowalark</creator><general>BioMed Central Ltd</general><general>BioMed Central</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20170823</creationdate><title>Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis</title><author>Mahittikorn, Aongart ; Thammasonthijarern, Nipa ; Roobthaisong, Amonrattana ; Udonsom, Ruenruetai ; Popruk, Supaluk ; Siri, Sukhontha ; Mori, Hirotake ; Sukthana, Yaowalark</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c500t-b963b9505398f8832c4616df7303b5dd4030c832a04b3677d4e2ad6e6bb8625f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Animals</topic><topic>Coccidians</topic><topic>Coccidiosis - diagnosis</topic><topic>Coccidiosis - parasitology</topic><topic>Coccidiosis - veterinary</topic><topic>Colorimetry</topic><topic>Coloring Agents - metabolism</topic><topic>DNA Primers</topic><topic>Dog Diseases - diagnosis</topic><topic>Dog Diseases - parasitology</topic><topic>Dogs</topic><topic>Feces - parasitology</topic><topic>Genes, Protozoan</topic><topic>Genetic aspects</topic><topic>Genetic testing</topic><topic>Genotypes</topic><topic>Host-parasite relationships</topic><topic>Innovations</topic><topic>Limit of Detection</topic><topic>Molecular Diagnostic Techniques</topic><topic>Neospora - genetics</topic><topic>Neospora - virology</topic><topic>Nucleic Acid Amplification Techniques - methods</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>Sensitivity and Specificity</topic><topic>Temperature</topic><topic>Thailand</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mahittikorn, Aongart</creatorcontrib><creatorcontrib>Thammasonthijarern, Nipa</creatorcontrib><creatorcontrib>Roobthaisong, Amonrattana</creatorcontrib><creatorcontrib>Udonsom, Ruenruetai</creatorcontrib><creatorcontrib>Popruk, Supaluk</creatorcontrib><creatorcontrib>Siri, Sukhontha</creatorcontrib><creatorcontrib>Mori, Hirotake</creatorcontrib><creatorcontrib>Sukthana, Yaowalark</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Parasites & vectors</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mahittikorn, Aongart</au><au>Thammasonthijarern, Nipa</au><au>Roobthaisong, Amonrattana</au><au>Udonsom, Ruenruetai</au><au>Popruk, Supaluk</au><au>Siri, Sukhontha</au><au>Mori, Hirotake</au><au>Sukthana, Yaowalark</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis</atitle><jtitle>Parasites & vectors</jtitle><addtitle>Parasit Vectors</addtitle><date>2017-08-23</date><risdate>2017</risdate><volume>10</volume><issue>1</issue><spage>394</spage><epage>394</epage><pages>394-394</pages><artnum>394</artnum><issn>1756-3305</issn><eissn>1756-3305</eissn><abstract>Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum.
LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand.
Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697).
This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.</abstract><cop>England</cop><pub>BioMed Central Ltd</pub><pmid>28835287</pmid><doi>10.1186/s13071-017-2330-2</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Springer Nature OA/Free Journals; SpringerLink Journals - AutoHoldings |
| subjects | Animals Coccidians Coccidiosis - diagnosis Coccidiosis - parasitology Coccidiosis - veterinary Colorimetry Coloring Agents - metabolism DNA Primers Dog Diseases - diagnosis Dog Diseases - parasitology Dogs Feces - parasitology Genes, Protozoan Genetic aspects Genetic testing Genotypes Host-parasite relationships Innovations Limit of Detection Molecular Diagnostic Techniques Neospora - genetics Neospora - virology Nucleic Acid Amplification Techniques - methods Real-Time Polymerase Chain Reaction - methods Sensitivity and Specificity Temperature Thailand |
| title | Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis |
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