Cargando…

Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples

Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-spec...

Descripción completa

Detalles Bibliográficos
Autores principales: Cisek, Agata Anna, Bąk, Iwona, Cukrowska, Bożena
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10051802/
https://www.ncbi.nlm.nih.gov/pubmed/36985233
http://dx.doi.org/10.3390/microorganisms11030660
_version_ 1785014977924431872
author Cisek, Agata Anna
Bąk, Iwona
Cukrowska, Bożena
author_facet Cisek, Agata Anna
Bąk, Iwona
Cukrowska, Bożena
author_sort Cisek, Agata Anna
collection PubMed
description Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples—or in this case, chicken droppings.
format Online
Article
Text
id pubmed-10051802
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-100518022023-03-30 Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples Cisek, Agata Anna Bąk, Iwona Cukrowska, Bożena Microorganisms Article Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples—or in this case, chicken droppings. MDPI 2023-03-05 /pmc/articles/PMC10051802/ /pubmed/36985233 http://dx.doi.org/10.3390/microorganisms11030660 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Cisek, Agata Anna
Bąk, Iwona
Cukrowska, Bożena
Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples
title Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples
title_full Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples
title_fullStr Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples
title_full_unstemmed Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples
title_short Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples
title_sort improved quantitative real-time pcr protocol for detection and quantification of methanogenic archaea in stool samples
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10051802/
https://www.ncbi.nlm.nih.gov/pubmed/36985233
http://dx.doi.org/10.3390/microorganisms11030660
work_keys_str_mv AT cisekagataanna improvedquantitativerealtimepcrprotocolfordetectionandquantificationofmethanogenicarchaeainstoolsamples
AT bakiwona improvedquantitativerealtimepcrprotocolfordetectionandquantificationofmethanogenicarchaeainstoolsamples
AT cukrowskabozena improvedquantitativerealtimepcrprotocolfordetectionandquantificationofmethanogenicarchaeainstoolsamples