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Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum
BACKGROUND: Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the ear...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099392/ https://www.ncbi.nlm.nih.gov/pubmed/24997675 http://dx.doi.org/10.1186/1756-0500-7-433 |
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author | Aguilera-Arreola, Ma Guadalupe González-Cardel, Ana María Tenorio, Alfonso Méndez Curiel-Quesada, Everardo Castro-Escarpulli, Graciela |
author_facet | Aguilera-Arreola, Ma Guadalupe González-Cardel, Ana María Tenorio, Alfonso Méndez Curiel-Quesada, Everardo Castro-Escarpulli, Graciela |
author_sort | Aguilera-Arreola, Ma Guadalupe |
collection | PubMed |
description | BACKGROUND: Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. METHODS: The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. RESULTS: Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 10(5), 3.9 × 10(3), 61.19 × 10(6) and 6.37 × 10(5) copies of a DNA template, respectively. CONCLUSIONS: The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. |
format | Online Article Text |
id | pubmed-4099392 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-40993922014-07-17 Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum Aguilera-Arreola, Ma Guadalupe González-Cardel, Ana María Tenorio, Alfonso Méndez Curiel-Quesada, Everardo Castro-Escarpulli, Graciela BMC Res Notes Research Article BACKGROUND: Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. METHODS: The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. RESULTS: Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 10(5), 3.9 × 10(3), 61.19 × 10(6) and 6.37 × 10(5) copies of a DNA template, respectively. CONCLUSIONS: The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. BioMed Central 2014-07-06 /pmc/articles/PMC4099392/ /pubmed/24997675 http://dx.doi.org/10.1186/1756-0500-7-433 Text en Copyright © 2014 Aguilera-Arreola et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Article Aguilera-Arreola, Ma Guadalupe González-Cardel, Ana María Tenorio, Alfonso Méndez Curiel-Quesada, Everardo Castro-Escarpulli, Graciela Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum |
title | Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum |
title_full | Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum |
title_fullStr | Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum |
title_full_unstemmed | Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum |
title_short | Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum |
title_sort | highly specific and efficient primers for in-house multiplex pcr detection of chlamydia trachomatis, neisseria gonorrhoeae, mycoplasma hominis and ureaplasma urealyticum |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099392/ https://www.ncbi.nlm.nih.gov/pubmed/24997675 http://dx.doi.org/10.1186/1756-0500-7-433 |
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