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

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 10(4)-10(10) D. 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 bq hybridization was 1 x 10(8) cells g(-1) VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 x 10(7) g(-1) VSS. This corresponds to a threshold level for hybridization of 0.1-0.001 parts per thousand of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0.01-0.0001 parts per thousand. 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.