Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides
Objective/background: The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl–Neelsen-stained slides (ZNS sl...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2015-01-01
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| Series: | International Journal of Mycobacteriology |
| Subjects: | |
| Online Access: | http://www.ijmyco.org/article.asp?issn=2212-5531;year=2015;volume=4;issue=4;spage=284;epage=289;aulast=Ruiz-Fuentes |
| _version_ | 1818309337716621312 |
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| author | Jenny Laura Ruiz-Fuentes Alexis Díaz Anayma Elena Entenza Yahima Frión Odelaisy Suárez Pedro Torres Yaxsier de Armas Lucrecia Acosta |
| author_facet | Jenny Laura Ruiz-Fuentes Alexis Díaz Anayma Elena Entenza Yahima Frión Odelaisy Suárez Pedro Torres Yaxsier de Armas Lucrecia Acosta |
| author_sort | Jenny Laura Ruiz-Fuentes |
| collection | DOAJ |
| description | Objective/background: The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl–Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit).
Methods: DNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]≥4), a low-codification-slide group (BI = 1), a negative-slide group (BI = 0), and a negative-control-slide group (BI = 0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction.
Results: This is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol–chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities.
Conclusion: The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques. |
| first_indexed | 2024-12-13T07:28:34Z |
| format | Article |
| id | doaj.art-b1ad590760504b34b1e9320058051586 |
| institution | Directory Open Access Journal |
| issn | 2212-5531 2212-554X |
| language | English |
| last_indexed | 2024-12-13T07:28:34Z |
| publishDate | 2015-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | International Journal of Mycobacteriology |
| spelling | doaj.art-b1ad590760504b34b1e93200580515862022-12-21T23:55:16ZengWolters Kluwer Medknow PublicationsInternational Journal of Mycobacteriology2212-55312212-554X2015-01-014428428910.1016/j.ijmyco.2015.06.005Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slidesJenny Laura Ruiz-FuentesAlexis DíazAnayma Elena EntenzaYahima FriónOdelaisy SuárezPedro TorresYaxsier de ArmasLucrecia AcostaObjective/background: The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl–Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit). Methods: DNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]≥4), a low-codification-slide group (BI = 1), a negative-slide group (BI = 0), and a negative-control-slide group (BI = 0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction. Results: This is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol–chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities. Conclusion: The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques.http://www.ijmyco.org/article.asp?issn=2212-5531;year=2015;volume=4;issue=4;spage=284;epage=289;aulast=Ruiz-FuentesDNA extraction methodsLeprosyMolecular diagnosisMycobacterium lepraeZiehl–Neelsen-stained microscopic slides |
| spellingShingle | Jenny Laura Ruiz-Fuentes Alexis Díaz Anayma Elena Entenza Yahima Frión Odelaisy Suárez Pedro Torres Yaxsier de Armas Lucrecia Acosta Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides International Journal of Mycobacteriology DNA extraction methods Leprosy Molecular diagnosis Mycobacterium leprae Ziehl–Neelsen-stained microscopic slides |
| title | Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides |
| title_full | Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides |
| title_fullStr | Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides |
| title_full_unstemmed | Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides |
| title_short | Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides |
| title_sort | comparison of four dna extraction methods for the detection of mycobacterium leprae from ziehl neelsen stained microscopic slides |
| topic | DNA extraction methods Leprosy Molecular diagnosis Mycobacterium leprae Ziehl–Neelsen-stained microscopic slides |
| url | http://www.ijmyco.org/article.asp?issn=2212-5531;year=2015;volume=4;issue=4;spage=284;epage=289;aulast=Ruiz-Fuentes |
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