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

Authors :
Agata Anna Cisek
Iwona Bąk
Bożena Cukrowska
Source :
Microorganisms, Vol 11, Iss 3, p 660 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

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.

Details

Language :
English
ISSN :
20762607 and 61146099
Volume :
11
Issue :
3
Database :
Directory of Open Access Journals
Journal :
Microorganisms
Publication Type :
Academic Journal
Accession number :
edsdoj.7771f8c4baf45d69b23af61146099b2
Document Type :
article
Full Text :
https://doi.org/10.3390/microorganisms11030660