Establishment Of Rapid Detection Techniques Of Two Strains Of Marine Probiotics And Their Applications

Marine bacteria Y5 which can degrade organics pollutants was isolated from healthy shrimp culture water and DL2 was isolated from microbial biofilms of antiseptic steel pieces in aquatic water which had antibacterial activity. Based on morphological, API 20E and 16S rRNA gene analysis, Y5 was identified as Bacillus firmus and DL2 was identified as Phaeobacter inhibens. Extracellular enzymatic activity of strain Y5 and inhibitory activity of strain DL2 were tested. The result showed that Y5 had high activities of caseinase, glutinase, amylase, lecithinase and lipase, and DL2 showed wide inhibitory spectrum in the 43 studied strains, especially for pathogenic Vibrio sp.Antiserum was produced in a female New Zealand white rabbit by injecting Y5. The indirect enzyme-linked immunosorbent assays (ELISA) developed for the rapid detection of Y5 was developed and the detection limit of the ELISA was 105 cell/ml.The gyrB genes of Y5 and DL2 were amplified using universal primers UP-1/UP-2r. Oligonucleotide PCR primers were designed based on the sequence of the target gyrB gene. The primer set BF1/BR2 can specifically detect Y5. The detection limit of primer BR1/BR2 was 7.6×103cfu/ml and in 50μl PCR mixtures the lower detection limits of primer BR1/BR2 was 12.5pg. The specific primer set PF1/PR2 can specifically detect DL2. The detection limit of primer PF1/PR2 was 2.98×104cfu/ml and in 50μl PCR mixtures the lower detection limits of primer PF1/PR2 was 31pg.Y5 and DL2 were applied in zebra fish and clam culture with high dose. The livability of Y5 and DL2 in host intestines was studied using PCR assay associated with plate count. Y5 remained viable more than 14 days in aquatic animals intestines, while DL2 remained viable for only about 24 h in the intestines. The results showed that Y5 can increase and survive rapidly in the host intestines, and the survival of DL2 was feeble in host intestines. The PCR assay was applied in the detection of samples isolated from different environment. For the detection of environmental total DNA of Qingdao seaside, no special bands were amplified with primer pairs BF1/BR2 and PF1/PR2. However, there were two positive isolates detected from environment with primer set BF1/BR2, while no positive isolates detected with primer set PF1/PR2. To ensure the detected veracity of primer BF1/BR2, 16S rRNA genes of the two positive isolates detected from aquatic environment were amplified and sequenced. The results showed that both of the positive isolates were identified as Bacillus firmus. In this study, it was found that there were more Bacillus firmus existed in aquatic environment than in other environment and there was few Phaeobacter inhibens existed in natural environment. Therefore, PCR amplification with the primer BF1/BR2 could be a rapid and reliable method for the detection of Bacillus firmus.