Design and application of a synthetic DNA standard for real-time PCR analysis of microbial communities in a biogas digester

Abstract

A synthetic DNA fragment containing primer binding sites for the quantification of ten different microbial groups was constructed and evaluated as a reliable enumeration standard for quantitative real-time PCR (qPCR) analyses. This approach has been exemplary verified for the quantification of several methanogenic orders and families in a series of samples drawn from a mesophilic biogas plant. Furthermore, the total amounts of bacteria as well as the number of sulfate-reducing and propionic acid bacteria as potential methanogenic interaction partners were successfully determined. The obtained results indicated a highly dynamic microbial community structure which was distinctly affected by the organic loading rate, the substrate selection, and the amount of free volatile fatty acids in the fermenter. Methanosarcinales was the most predominant methanogenic order during the 3 months of observation despite fluctuating process conditions. During all trials, the modified quantification standard indicated a maximum of reproducibility and efficiency, enabling this method to open up a wide range of novel application options.

Analysis of propionate‐degrading consortia from agricultural biogas plants

“…Quantification of total bacteria and total archaea was determined according to (May et al., ), using an artificial DNA fragment for standard preparation and the primer combinations BAC338F/BAC805R and 931F/M1100R for bacterial and archaeal 16S rRNA gene‐fragment amplification. The qPCR assays were performed using a realplex2 ep gradient S Mastercycler (Eppendorf AG, Hamburg, Germany) supported by the evaluation software realplex 2.2.…”

Online monitoring of stable carbon isotopes of methane in anaerobic digestion as a new tool for early warning of process instability

“…PAB were identified by oligomers that bind to the propionate-CoA transferase gene sequence (pct) (Li et al, 2013). A synthetic DNA strand con- taining the binding sites for each primer pair of this study was exerted as copy number standard (May et al, 2015). QPCR approaches were carried out as SYBR Ò Green assays using a realplex 2 ep gradient S Mastercycler (Eppendorf AG, Hamburg, Germany).…”

“…Bacterial and methanogenic community structures were monitored by qPCR analyses as previously described (May et al, 2015).…”

Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes

“…For instance, both PCR amplicons and plasmids need to be purified before being used, procedure which is often causing contaminations ( Cimino et al, 1991 ). Moreover, the quantification of plasmid copies per cell was shown to be unreliable ( May et al, 2015 ; Conte et al, 2018 ). In recent years, there has been a growing interest to use artificially synthesized DNA and RNA sequences as qPCR standards.…”

“…Synthesizing such sequences to produce standards is considerably faster, cleaner (low contamination risk) and also less expensive (following considerable reduction of the cost of custom DNA synthesis over the years, see Carlson, 2009 ) compared to traditional plasmid standards ( Conte et al, 2018 ; Xu et al, 2019 ). The synthetic gene fragments can be purchased in a length of 125 to 3,000 base pair (bp) with known degenerate nucleotides of A, T, C, and G ( May et al, 2015 ; Conte et al, 2018 ). Up to now, most of the artificially synthesized standards have been used for medical purpose, focusing on viral or infectious microorganisms ( Tourinho et al, 2015 ; Fesolovich and Tobe, 2017 ; Lima et al, 2017 ; Magee et al, 2017 ; Bandeira et al, 2020 ; Bivins et al, 2021 ; Munoz-Calderon et al, 2021 ), very few in environmental samples.…”