Kuo Testing Laboratories, Othello, WA, 99344, USA
Development of Methanoculleus-specific real-time quantitative PCR assay for assessing methanogen communities in anaerobic digestion
Article first published online: 4 MAR 2014
© 2014 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 116, Issue 6, pages 1474–1481, June 2014
How to Cite
Chen, S., Zhu, Z., Park, J., Zhang, Z. and He, Q. (2014), Development of Methanoculleus-specific real-time quantitative PCR assay for assessing methanogen communities in anaerobic digestion. Journal of Applied Microbiology, 116: 1474–1481. doi: 10.1111/jam.12471
- Issue published online: 19 MAY 2014
- Article first published online: 4 MAR 2014
- Accepted manuscript online: 12 FEB 2014 11:31PM EST
- Manuscript Accepted: 3 FEB 2014
- Manuscript Revised: 8 DEC 2013
- Manuscript Received: 7 SEP 2013
- Science Alliance—Tennessee Center of Excellence
- US Environmental Protection Agency. Grant Number: SU834318
- Institute for a Secure and Sustainable Environment
- Pre-Collegiate Research Scholars program at the University of Tennessee
- anaerobic digestion;
- Methanoculleus ;
- quantitative PCR;
- real-time PCR
To develop a Methanoculleus-specific real-time quantitative PCR (RT-qPCR) assay with high coverage and specificity for the analysis of methanogenic populations in anaerobic digestion.
Methods and Results
A Methanoculleus-specific primer/probe set for RT-qPCR was designed in this study based on all Methanoculleus 16S rRNA gene sequences in Ribosomal Database Project (RDP) according to TaqMan chemistry. The newly designed primer/probe set was shown to have high coverage and specificity by both in silico and experimental analyses. Amplification efficiency of the Methanoculleus-specific primer/probe set was determined to be ideal for RT-qPCR applications. Subsequent field testing on anaerobic digesters showed that results from RT-qPCR were consistent with those from clone library analysis, validating the accuracy of the RT-qPCR assay.
The Methanoculleus-specific RT-qPCR assay designed in this study can serve as a rapid and effective tool for the quantification of Methanoculleus populations in anaerobic digestion.
Significance and Impact of the Study
Methanoculleus populations represent important members of archaeal communities in methanogenic processes, necessitating the need to develop effective tools to monitor Methanoculleus population abundance. The RT-qPCR developed in this study provides an essential tool for the quantification of Methanoculleus populations in anaerobic digestion and for the understanding of the functions of these methanogens in anaerobic biotransformation.