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Development of M ethanoculleus ‐specific real‐time quantitative PCR assay for assessing methanogen communities in anaerobic digestion
Author(s) -
Chen S.,
Zhu Z.,
Park J.,
Zhang Z.,
He Q.
Publication year - 2014
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12471
Subject(s) - library science , zhàng , political science , china , law , computer science
Aim To develop a M ethanoculleus ‐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 M ethanoculleus ‐specific primer/probe set for RT ‐ qPCR was designed in this study based on all M ethanoculleus 16 S rRNA gene sequences in Ribosomal Database Project ( RDP ) according to T aq M an 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 M ethanoculleus ‐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. Conclusions The M ethanoculleus ‐specific RT ‐ qPCR assay designed in this study can serve as a rapid and effective tool for the quantification of M ethanoculleus populations in anaerobic digestion. Significance and Impact of the Study M ethanoculleus populations represent important members of archaeal communities in methanogenic processes, necessitating the need to develop effective tools to monitor M ethanoculleus population abundance. The RT ‐ qPCR developed in this study provides an essential tool for the quantification of M ethanoculleus populations in anaerobic digestion and for the understanding of the functions of these methanogens in anaerobic biotransformation.