Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products
We developed a protocol for detection of mutations in the pncA gene associated with M. tuberculosis resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared i...
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| Veröffentlicht in: | Bulletin of experimental biology and medicine 2019-12, Vol.168 (2), p.264-269 |
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| creator | Filipenko, M. L. Dymova, M. A. Cherednichenko, A. G. Khrapov, E. A. Mishukova, O. V. Schwartz, Ya. Sh |
| description | We developed a protocol for detection of mutations in the
pncA
gene associated with
M. tuberculosis
resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZA
R
DNA isolates carrying mutations in the
pncA
gene, 3 PZA
R
isolates without mutations in the
pncA
gene, and 20 PZA
S
isolates without mutations in the
pncA
gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the
pncA
gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy — 96.55%
vs.
93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software. |
| doi_str_mv | 10.1007/s10517-019-04688-6 |
| format | Article |
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pncA
gene associated with
M. tuberculosis
resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZA
R
DNA isolates carrying mutations in the
pncA
gene, 3 PZA
R
isolates without mutations in the
pncA
gene, and 20 PZA
S
isolates without mutations in the
pncA
gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the
pncA
gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy — 96.55%
vs.
93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software.</description><identifier>ISSN: 0007-4888</identifier><identifier>EISSN: 1573-8221</identifier><identifier>DOI: 10.1007/s10517-019-04688-6</identifier><identifier>PMID: 31782002</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Amidohydrolases - genetics ; Antitubercular Agents - pharmacology ; Base Sequence ; Biomedical and Life Sciences ; Biomedicine ; Cell Biology ; Comparative analysis ; Drug resistance in microorganisms ; Drug Resistance, Bacterial - genetics ; Genes ; Genetic aspects ; Internal Medicine ; Laboratory Medicine ; Mycobacterium tuberculosis - drug effects ; Mycobacterium tuberculosis - genetics ; Mycobacterium tuberculosis - isolation & purification ; Nucleic Acid Denaturation - genetics ; Pathology ; Polymorphism, Single Nucleotide - genetics ; Pyrazinamide ; Pyrazinamide - pharmacology ; Sequence Analysis, DNA ; Tuberculosis ; Tuberculosis, Pulmonary - drug therapy ; Tuberculosis, Pulmonary - microbiology</subject><ispartof>Bulletin of experimental biology and medicine, 2019-12, Vol.168 (2), p.264-269</ispartof><rights>Springer Science+Business Media, LLC, part of Springer Nature 2019</rights><rights>COPYRIGHT 2019 Springer</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c445t-a758a02ebf5e36479d85db21ca68b8e5956a8bb5d1380bcb84a4ab2de86666a63</citedby><cites>FETCH-LOGICAL-c445t-a758a02ebf5e36479d85db21ca68b8e5956a8bb5d1380bcb84a4ab2de86666a63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10517-019-04688-6$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10517-019-04688-6$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>315,781,785,27929,27930,41493,42562,51324</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31782002$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Filipenko, M. L.</creatorcontrib><creatorcontrib>Dymova, M. A.</creatorcontrib><creatorcontrib>Cherednichenko, A. G.</creatorcontrib><creatorcontrib>Khrapov, E. A.</creatorcontrib><creatorcontrib>Mishukova, O. V.</creatorcontrib><creatorcontrib>Schwartz, Ya. Sh</creatorcontrib><title>Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products</title><title>Bulletin of experimental biology and medicine</title><addtitle>Bull Exp Biol Med</addtitle><addtitle>Bull Exp Biol Med</addtitle><description>We developed a protocol for detection of mutations in the
pncA
gene associated with
M. tuberculosis
resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZA
R
DNA isolates carrying mutations in the
pncA
gene, 3 PZA
R
isolates without mutations in the
pncA
gene, and 20 PZA
S
isolates without mutations in the
pncA
gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the
pncA
gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy — 96.55%
vs.
93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software.</description><subject>Amidohydrolases - genetics</subject><subject>Antitubercular Agents - pharmacology</subject><subject>Base Sequence</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Cell Biology</subject><subject>Comparative analysis</subject><subject>Drug resistance in microorganisms</subject><subject>Drug Resistance, Bacterial - genetics</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Internal Medicine</subject><subject>Laboratory Medicine</subject><subject>Mycobacterium tuberculosis - drug effects</subject><subject>Mycobacterium tuberculosis - genetics</subject><subject>Mycobacterium tuberculosis - isolation & purification</subject><subject>Nucleic Acid Denaturation - genetics</subject><subject>Pathology</subject><subject>Polymorphism, Single Nucleotide - genetics</subject><subject>Pyrazinamide</subject><subject>Pyrazinamide - pharmacology</subject><subject>Sequence Analysis, DNA</subject><subject>Tuberculosis</subject><subject>Tuberculosis, Pulmonary - drug therapy</subject><subject>Tuberculosis, Pulmonary - microbiology</subject><issn>0007-4888</issn><issn>1573-8221</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kV9vFCEUxYnR2LX6BXwwJCamL1OBmWHYx81qW5NubBp9JsDc2aWZhZU_JvvoN5fptMYmRnjgAr9zyOUg9JaSc0pI9zFS0tKuInRZkYYLUfFnaEHbrq4EY_Q5WpBCVY0Q4gS9ivFu2hJOX6KTmnaCEcIW6NcnSGCS9Q77AW9yUlMdsXV4czReK5Mg2LzHKWsIJo8-2ogPzqzwJTjA-og3vreDhR5f2e2uuoXox3xvuIExWbfF6xx-Al45NR4ncXnnZn2Lb4Lvs0nxNXoxqDHCm4f1FH2_-PxtfVVdf738sl5dV6Zp2lSprhWKMNBDCzVvumUv2l4zahQXWkC7bLkSWrc9rQXRRotGNUqzHgQvQ_H6FJ3Nvofgf2SISe5tNDCOyoHPUbKakbrrCKEFfT-jWzWCtG7wKSgz4XLFacuXlJGmUOf_oMrsYW-NdzDYcv5E8OEvwQ7UmHaPnxWfgmwGTfAxBhjkIdi9CkdJiZyil3P0skQv76OXU3_vHvrLeg_9H8lj1gWoZyCWK7eFIO98DiWV-D_b3xHJuJc</recordid><startdate>20191201</startdate><enddate>20191201</enddate><creator>Filipenko, M. L.</creator><creator>Dymova, M. A.</creator><creator>Cherednichenko, A. G.</creator><creator>Khrapov, E. A.</creator><creator>Mishukova, O. V.</creator><creator>Schwartz, Ya. Sh</creator><general>Springer US</general><general>Springer</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>7X8</scope></search><sort><creationdate>20191201</creationdate><title>Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products</title><author>Filipenko, M. L. ; Dymova, M. A. ; Cherednichenko, A. G. ; Khrapov, E. A. ; Mishukova, O. V. ; Schwartz, Ya. Sh</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c445t-a758a02ebf5e36479d85db21ca68b8e5956a8bb5d1380bcb84a4ab2de86666a63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Amidohydrolases - genetics</topic><topic>Antitubercular Agents - pharmacology</topic><topic>Base Sequence</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Cell Biology</topic><topic>Comparative analysis</topic><topic>Drug resistance in microorganisms</topic><topic>Drug Resistance, Bacterial - genetics</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Internal Medicine</topic><topic>Laboratory Medicine</topic><topic>Mycobacterium tuberculosis - drug effects</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>Mycobacterium tuberculosis - isolation & purification</topic><topic>Nucleic Acid Denaturation - genetics</topic><topic>Pathology</topic><topic>Polymorphism, Single Nucleotide - genetics</topic><topic>Pyrazinamide</topic><topic>Pyrazinamide - pharmacology</topic><topic>Sequence Analysis, DNA</topic><topic>Tuberculosis</topic><topic>Tuberculosis, Pulmonary - drug therapy</topic><topic>Tuberculosis, Pulmonary - microbiology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Filipenko, M. L.</creatorcontrib><creatorcontrib>Dymova, M. A.</creatorcontrib><creatorcontrib>Cherednichenko, A. G.</creatorcontrib><creatorcontrib>Khrapov, E. A.</creatorcontrib><creatorcontrib>Mishukova, O. V.</creatorcontrib><creatorcontrib>Schwartz, Ya. Sh</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Bulletin of experimental biology and medicine</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Filipenko, M. L.</au><au>Dymova, M. A.</au><au>Cherednichenko, A. G.</au><au>Khrapov, E. A.</au><au>Mishukova, O. V.</au><au>Schwartz, Ya. Sh</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products</atitle><jtitle>Bulletin of experimental biology and medicine</jtitle><stitle>Bull Exp Biol Med</stitle><addtitle>Bull Exp Biol Med</addtitle><date>2019-12-01</date><risdate>2019</risdate><volume>168</volume><issue>2</issue><spage>264</spage><epage>269</epage><pages>264-269</pages><issn>0007-4888</issn><eissn>1573-8221</eissn><abstract>We developed a protocol for detection of mutations in the
pncA
gene associated with
M. tuberculosis
resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZA
R
DNA isolates carrying mutations in the
pncA
gene, 3 PZA
R
isolates without mutations in the
pncA
gene, and 20 PZA
S
isolates without mutations in the
pncA
gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the
pncA
gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy — 96.55%
vs.
93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>31782002</pmid><doi>10.1007/s10517-019-04688-6</doi><tpages>6</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0007-4888 |
| ispartof | Bulletin of experimental biology and medicine, 2019-12, Vol.168 (2), p.264-269 |
| issn | 0007-4888 1573-8221 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_2320377001 |
| source | MEDLINE; SpringerNature Journals |
| subjects | Amidohydrolases - genetics Antitubercular Agents - pharmacology Base Sequence Biomedical and Life Sciences Biomedicine Cell Biology Comparative analysis Drug resistance in microorganisms Drug Resistance, Bacterial - genetics Genes Genetic aspects Internal Medicine Laboratory Medicine Mycobacterium tuberculosis - drug effects Mycobacterium tuberculosis - genetics Mycobacterium tuberculosis - isolation & purification Nucleic Acid Denaturation - genetics Pathology Polymorphism, Single Nucleotide - genetics Pyrazinamide Pyrazinamide - pharmacology Sequence Analysis, DNA Tuberculosis Tuberculosis, Pulmonary - drug therapy Tuberculosis, Pulmonary - microbiology |
| title | Detection of Mutations in Mycobacterium tuberculosis pncA Gene by Modified High-Resolution Melting Curve Analysis of PCR Products |
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