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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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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 |
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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 |
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