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Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens
BACKGROUND & OBJECTIVES: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - stain...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Medknow Publications & Media Pvt Ltd
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248388/ https://www.ncbi.nlm.nih.gov/pubmed/25366209 |
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author | Revathy, Mani Therese, Kulandai Lily Bagyalakshmi, Radhakishnan Chandrasekar, Chokaliingam Kumar, Suria Madhavan, Hajib N. |
author_facet | Revathy, Mani Therese, Kulandai Lily Bagyalakshmi, Radhakishnan Chandrasekar, Chokaliingam Kumar, Suria Madhavan, Hajib N. |
author_sort | Revathy, Mani |
collection | PubMed |
description | BACKGROUND & OBJECTIVES: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients. METHODS: One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis. RESULTS: Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank. INTERPRETATION & CONCLUSIONS: Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection. |
format | Online Article Text |
id | pubmed-4248388 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Medknow Publications & Media Pvt Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-42483882014-12-05 Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens Revathy, Mani Therese, Kulandai Lily Bagyalakshmi, Radhakishnan Chandrasekar, Chokaliingam Kumar, Suria Madhavan, Hajib N. Indian J Med Res Original Article BACKGROUND & OBJECTIVES: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients. METHODS: One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis. RESULTS: Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank. INTERPRETATION & CONCLUSIONS: Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection. Medknow Publications & Media Pvt Ltd 2014-09 /pmc/articles/PMC4248388/ /pubmed/25366209 Text en Copyright: © Indian Journal of Medical Research http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Revathy, Mani Therese, Kulandai Lily Bagyalakshmi, Radhakishnan Chandrasekar, Chokaliingam Kumar, Suria Madhavan, Hajib N. Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens |
title | Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens |
title_full | Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens |
title_fullStr | Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens |
title_full_unstemmed | Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens |
title_short | Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens |
title_sort | application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of pneumocystis jirovecii in clinical specimens |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248388/ https://www.ncbi.nlm.nih.gov/pubmed/25366209 |
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