Cisek Agata Anna, Bąk Iwona, Cukrowska Bożena
Department of Pathology, The Children's Memorial Health Institute, Av. Dzieci Polskich 20, 04-730 Warsaw, Poland.
Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, St. Ciszewskiego 8, 02-786 Warsaw, Poland.
Microorganisms. 2023 Mar 5;11(3):660. doi: 10.3390/microorganisms11030660.
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 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 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.
产甲烷古菌是人和动物肠道微生物群的重要组成部分，但在描述该主题的出版物中却很少报道它们的存在。定量产甲烷菌流行率的方法之一是对产甲烷菌特异性基因进行定量实时PCR（qPCR），检测失败的一个可能原因通常是方法偏差。在这里，我们通过更换其中一个引物并改善qPCR反应条件来改进现有方案。结果，以略低但可接受的PCR效率为代价，新检测方法具有更高的特异性和灵敏度，线性检测范围扩大到7个数量级。在100%的频率下定量的最低拷贝数为每个反应21个拷贝。测试的其他验证参数，如重复性和线性，也给出了令人满意的结果。总体而言，我们能够将引物二聚体化和其他交叉反应对qPCR的负面影响降至最低，并增加不仅可检测而且可定量的粪便样本数量——在这种情况下是鸡粪。
