Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

• Published in: Journal of applied microbiology 1997-07, Vol.83 (1), p.102-110
• Main Authors: Oude Elferink, S.J.W.H., Rinia, H.A., Bruins, M.E., De Vos, W.M., Stams, A.J.M.
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Microbiology
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1046/j.1365-2672.1997.00197.x

A species‐specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 104–1010D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell−1, the lowest number of target cells detected by hybridization was 1 × 108 cells g−1 VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 107 g−1 VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell−1 in slow‐growing cultures to 90 fg cell−1 in fast‐growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast‐growing cells is lower than for slow‐growing cells.