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Improved DNA extraction technique from clot for the diagnosis of Chagas disease
The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA...
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| Published in: | PLoS neglected tropical diseases 2019-01, Vol.13 (1), p.e0007024 |
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| creator | Mayta, Holger Romero, Yomara K Pando, Alejandra Verastegui, Manuela Tinajeros, Freddy Bozo, Ricardo Henderson-Frost, Josephine Colanzi, Rony Flores, Jorge Lerner, Richard Bern, Caryn Gilman, Robert H |
| description | The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease.
A total of 265 match pair samples of whole blood-guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.
The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease. |
| doi_str_mv | 10.1371/journal.pntd.0007024 |
| format | article |
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A total of 265 match pair samples of whole blood-guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.
The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.</description><identifier>ISSN: 1935-2735</identifier><identifier>ISSN: 1935-2727</identifier><identifier>EISSN: 1935-2735</identifier><identifier>DOI: 10.1371/journal.pntd.0007024</identifier><identifier>PMID: 30633743</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Biology and Life Sciences ; Blood ; Blood clots ; Buffy coat ; Cardiomyopathy ; Chagas disease ; Chagas Disease - diagnosis ; Chagas Disease - parasitology ; Chloroform ; Clinical trials ; Congenital diseases ; Deoxyribonucleic acid ; Detection ; Diagnosis ; Diagnostic software ; Diagnostic systems ; Diagnostic Tests, Routine - methods ; Diseases ; DNA ; DNA, Protozoan - blood ; DNA, Protozoan - genetics ; Genetic research ; Guanidine ; Hospitals ; Humans ; Infectious diseases ; Laboratories ; Medical schools ; Medicine and Health Sciences ; Methodology ; Methods ; Molecular Diagnostic Techniques ; Nucleotide sequence ; Parasites ; PCR ; Phenols ; Phenols (Class of compounds) ; Philosophy ; Public health ; Real-Time Polymerase Chain Reaction - methods ; Research and analysis methods ; Samples ; Sensitivity and Specificity ; Serologic Tests - methods ; Serology ; Systematic review ; Tropical diseases ; Trypanosoma ; Trypanosoma cruzi - genetics ; Trypanosoma cruzi - isolation & purification ; Vector-borne diseases ; Working groups</subject><ispartof>PLoS neglected tropical diseases, 2019-01, Vol.13 (1), p.e0007024</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Mayta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 Mayta et al 2019 Mayta et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c624t-7a64a3705f649240726e518a5ca7b7e1f011e7e4e8d8964750bf64f374651f033</citedby><cites>FETCH-LOGICAL-c624t-7a64a3705f649240726e518a5ca7b7e1f011e7e4e8d8964750bf64f374651f033</cites><orcidid>0000-0001-5306-628X ; 0000-0002-0429-8719</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2262870484/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2262870484?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30633743$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Nagarkatti, Rana</contributor><creatorcontrib>Mayta, Holger</creatorcontrib><creatorcontrib>Romero, Yomara K</creatorcontrib><creatorcontrib>Pando, Alejandra</creatorcontrib><creatorcontrib>Verastegui, Manuela</creatorcontrib><creatorcontrib>Tinajeros, Freddy</creatorcontrib><creatorcontrib>Bozo, Ricardo</creatorcontrib><creatorcontrib>Henderson-Frost, Josephine</creatorcontrib><creatorcontrib>Colanzi, Rony</creatorcontrib><creatorcontrib>Flores, Jorge</creatorcontrib><creatorcontrib>Lerner, Richard</creatorcontrib><creatorcontrib>Bern, Caryn</creatorcontrib><creatorcontrib>Gilman, Robert H</creatorcontrib><creatorcontrib>Chagas Working Group in Perú and Bolivia</creatorcontrib><creatorcontrib>for the Chagas Working Group in Perú and Bolivia</creatorcontrib><title>Improved DNA extraction technique from clot for the diagnosis of Chagas disease</title><title>PLoS neglected tropical diseases</title><addtitle>PLoS Negl Trop Dis</addtitle><description>The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease.
A total of 265 match pair samples of whole blood-guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.
The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.</description><subject>Biology and Life Sciences</subject><subject>Blood</subject><subject>Blood clots</subject><subject>Buffy coat</subject><subject>Cardiomyopathy</subject><subject>Chagas disease</subject><subject>Chagas Disease - diagnosis</subject><subject>Chagas Disease - parasitology</subject><subject>Chloroform</subject><subject>Clinical trials</subject><subject>Congenital diseases</subject><subject>Deoxyribonucleic acid</subject><subject>Detection</subject><subject>Diagnosis</subject><subject>Diagnostic software</subject><subject>Diagnostic systems</subject><subject>Diagnostic Tests, Routine - methods</subject><subject>Diseases</subject><subject>DNA</subject><subject>DNA, Protozoan - blood</subject><subject>DNA, Protozoan - genetics</subject><subject>Genetic research</subject><subject>Guanidine</subject><subject>Hospitals</subject><subject>Humans</subject><subject>Infectious diseases</subject><subject>Laboratories</subject><subject>Medical schools</subject><subject>Medicine and Health Sciences</subject><subject>Methodology</subject><subject>Methods</subject><subject>Molecular Diagnostic Techniques</subject><subject>Nucleotide sequence</subject><subject>Parasites</subject><subject>PCR</subject><subject>Phenols</subject><subject>Phenols (Class of compounds)</subject><subject>Philosophy</subject><subject>Public health</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Research and analysis methods</subject><subject>Samples</subject><subject>Sensitivity and Specificity</subject><subject>Serologic Tests - methods</subject><subject>Serology</subject><subject>Systematic review</subject><subject>Tropical diseases</subject><subject>Trypanosoma</subject><subject>Trypanosoma cruzi - genetics</subject><subject>Trypanosoma cruzi - isolation & purification</subject><subject>Vector-borne diseases</subject><subject>Working 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DNA extraction technique from clot for the diagnosis of Chagas disease</title><author>Mayta, Holger ; Romero, Yomara K ; Pando, Alejandra ; Verastegui, Manuela ; Tinajeros, Freddy ; Bozo, Ricardo ; Henderson-Frost, Josephine ; Colanzi, Rony ; Flores, Jorge ; Lerner, Richard ; Bern, Caryn ; Gilman, Robert H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c624t-7a64a3705f649240726e518a5ca7b7e1f011e7e4e8d8964750bf64f374651f033</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Biology and Life Sciences</topic><topic>Blood</topic><topic>Blood clots</topic><topic>Buffy coat</topic><topic>Cardiomyopathy</topic><topic>Chagas disease</topic><topic>Chagas Disease - diagnosis</topic><topic>Chagas Disease - parasitology</topic><topic>Chloroform</topic><topic>Clinical trials</topic><topic>Congenital diseases</topic><topic>Deoxyribonucleic 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tropical diseases</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mayta, Holger</au><au>Romero, Yomara K</au><au>Pando, Alejandra</au><au>Verastegui, Manuela</au><au>Tinajeros, Freddy</au><au>Bozo, Ricardo</au><au>Henderson-Frost, Josephine</au><au>Colanzi, Rony</au><au>Flores, Jorge</au><au>Lerner, Richard</au><au>Bern, Caryn</au><au>Gilman, Robert H</au><au>Nagarkatti, Rana</au><aucorp>Chagas Working Group in Perú and Bolivia</aucorp><aucorp>for the Chagas Working Group in Perú and Bolivia</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Improved DNA extraction technique from clot for the diagnosis of Chagas disease</atitle><jtitle>PLoS neglected tropical diseases</jtitle><addtitle>PLoS Negl Trop Dis</addtitle><date>2019-01-01</date><risdate>2019</risdate><volume>13</volume><issue>1</issue><spage>e0007024</spage><pages>e0007024-</pages><issn>1935-2735</issn><issn>1935-2727</issn><eissn>1935-2735</eissn><abstract>The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease.
A total of 265 match pair samples of whole blood-guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.
The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>30633743</pmid><doi>10.1371/journal.pntd.0007024</doi><orcidid>https://orcid.org/0000-0001-5306-628X</orcidid><orcidid>https://orcid.org/0000-0002-0429-8719</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1935-2735 |
| ispartof | PLoS neglected tropical diseases, 2019-01, Vol.13 (1), p.e0007024 |
| issn | 1935-2735 1935-2727 1935-2735 |
| language | eng |
| recordid | cdi_plos_journals_2262870484 |
| source | Publicly Available Content Database (Proquest) (PQ_SDU_P3); NCBI_PubMed Central（免费） |
| subjects | Biology and Life Sciences Blood Blood clots Buffy coat Cardiomyopathy Chagas disease Chagas Disease - diagnosis Chagas Disease - parasitology Chloroform Clinical trials Congenital diseases Deoxyribonucleic acid Detection Diagnosis Diagnostic software Diagnostic systems Diagnostic Tests, Routine - methods Diseases DNA DNA, Protozoan - blood DNA, Protozoan - genetics Genetic research Guanidine Hospitals Humans Infectious diseases Laboratories Medical schools Medicine and Health Sciences Methodology Methods Molecular Diagnostic Techniques Nucleotide sequence Parasites PCR Phenols Phenols (Class of compounds) Philosophy Public health Real-Time Polymerase Chain Reaction - methods Research and analysis methods Samples Sensitivity and Specificity Serologic Tests - methods Serology Systematic review Tropical diseases Trypanosoma Trypanosoma cruzi - genetics Trypanosoma cruzi - isolation & purification Vector-borne diseases Working groups |
| title | Improved DNA extraction technique from clot for the diagnosis of Chagas disease |
| url | http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-06T09%3A36%3A14IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Improved%20DNA%20extraction%20technique%20from%20clot%20for%20the%20diagnosis%20of%20Chagas%20disease&rft.jtitle=PLoS%20neglected%20tropical%20diseases&rft.au=Mayta,%20Holger&rft.aucorp=Chagas%20Working%20Group%20in%20Per%C3%BA%20and%20Bolivia&rft.date=2019-01-01&rft.volume=13&rft.issue=1&rft.spage=e0007024&rft.pages=e0007024-&rft.issn=1935-2735&rft.eissn=1935-2735&rft_id=info:doi/10.1371/journal.pntd.0007024&rft_dat=%3Cgale_plos_%3EA571883871%3C/gale_plos_%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c624t-7a64a3705f649240726e518a5ca7b7e1f011e7e4e8d8964750bf64f374651f033%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=2262870484&rft_id=info:pmid/30633743&rft_galeid=A571883871&rfr_iscdi=true |