Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction
BACKGROUND: Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Thieme Medical and Scientific Publishers Pvt. Ltd.
2018-01-01
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| Series: | Journal of Laboratory Physicians |
| Subjects: | |
| Online Access: | http://www.thieme-connect.de/DOI/DOI?10.4103/JLP.JLP_74_17 |
| _version_ | 1818160892324347904 |
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| author | Raghavendra D. Kulkarni Mukti Nath Mishra Jeevanandam Mohanraj Arun Chandrasekhar G S. Ajantha Sheetal Kulkani Shama Bhat |
| author_facet | Raghavendra D. Kulkarni Mukti Nath Mishra Jeevanandam Mohanraj Arun Chandrasekhar G S. Ajantha Sheetal Kulkani Shama Bhat |
| author_sort | Raghavendra D. Kulkarni |
| collection | DOAJ |
| description | BACKGROUND: Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis.
AIM AND OBJECTIVE: The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species.
MATERIALS AND METHODS: Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test.
RESULTS: The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells.
CONCLUSION: Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor. |
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| format | Article |
| id | doaj.art-9f6a51aa2fa24c1da8e271d11392b9f6 |
| institution | Directory Open Access Journal |
| issn | 0974-2727 0974-7826 |
| language | English |
| last_indexed | 2024-12-11T16:09:06Z |
| publishDate | 2018-01-01 |
| publisher | Thieme Medical and Scientific Publishers Pvt. Ltd. |
| record_format | Article |
| series | Journal of Laboratory Physicians |
| spelling | doaj.art-9f6a51aa2fa24c1da8e271d11392b9f62022-12-22T00:59:07ZengThieme Medical and Scientific Publishers Pvt. Ltd.Journal of Laboratory Physicians0974-27270974-78262018-01-01100106807210.4103/JLP.JLP_74_17Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reactionRaghavendra D. Kulkarni0Mukti Nath Mishra1Jeevanandam Mohanraj2Arun Chandrasekhar3G S. Ajantha4Sheetal Kulkani5Shama Bhat6Department of Molecular Diagnostics, Bhat Biotech Pvt. Ltd., Bengaluru, Karnataka, IndiaDepartment of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad,IndiaDepartment of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad,IndiaDepartment of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad,IndiaDepartment of Molecular Diagnostics, Bhat Biotech Pvt. Ltd., Bengaluru, Karnataka, IndiaDepartment of Molecular Diagnostics, Bhat Biotech Pvt. Ltd., Bengaluru, Karnataka, IndiaDepartment of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad,IndiaBACKGROUND: Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis. AIM AND OBJECTIVE: The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species. MATERIALS AND METHODS: Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test. RESULTS: The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells. CONCLUSION: Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor.http://www.thieme-connect.de/DOI/DOI?10.4103/JLP.JLP_74_17 acinetobacter dnadry-reagent mixinhibitorspolymerase chain reaction |
| spellingShingle | Raghavendra D. Kulkarni Mukti Nath Mishra Jeevanandam Mohanraj Arun Chandrasekhar G S. Ajantha Sheetal Kulkani Shama Bhat Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction Journal of Laboratory Physicians acinetobacter dna dry-reagent mix inhibitors polymerase chain reaction |
| title | Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction |
| title_full | Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction |
| title_fullStr | Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction |
| title_full_unstemmed | Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction |
| title_short | Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction |
| title_sort | development of a dry reagent mix based polymerase chain reaction as a novel tool for the identification of acinetobacter species and its comparison with conventional polymerase chain reaction |
| topic | acinetobacter dna dry-reagent mix inhibitors polymerase chain reaction |
| url | http://www.thieme-connect.de/DOI/DOI?10.4103/JLP.JLP_74_17 |
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