Study On Optimize Detection Technology In Exogenous Lactobacillus Of Fish Gut And Metabolic Study

Objective: For studying the metabolism of exogenous lactobacillus in the gut of tilapia and zebrafish by using quantitative PCR, the objective of this study was to optimize the extraction process of microorganism genomic DNA from fish gut. With the purpose to screen Lactobacillus strains with high colonization in fish gut, the effect of dose and duration on the colonization of Lactobacillus in fish intestine was investigated subsequently.Method: Tilapia(1.5- 2.5 g) and TU zebrafish(0.3-0.5 g) were used to compare the effect of different sample-pretreatment methods, CTAB method and DNA extraction Kit on extraction rate and quality of extracted DNA from intestinal microorganism. The CTAB protocol was adjusted to improve the extraction rate and quality of extracted DNA. The levels of orally supplemented LGG, 1170 and JCM1149 in fish gut was detected using qPCR with specific primers for each strain. The detection limit of qPCR was evaluated by conducting the same qPCR procedures on DNA extracted from fish intestine sample with no exogenous Lactobacillus addition. The precision in detecting JCM1149 and LGG of the optimized qPCR method was compared with that of the plate count method. Lastly, we investigated the metabolism of orally applied JCM1149 in intestine of tilapia and zebrafish by the optimized qPCR detection technique, including evacuation analysis of JCM1149 after 7 days, and saturation analysis of JCM1149 after 3 days with four concentration gradients of JCM1149 during immersion bathing of fish.Result:(1) The substances that influence subsequent DNA extraction and qPCR reaction can be eliminated by PEG8000 addition after homogenization followed by centrifugation at 1000 rpm for 10 min. Results of plate counting indicated that the supernatant retained more than 90% of the bacteria from the pre-treatment samples. Moreover, removal of impurities greatly reduced the background value of samples.(2) The purity of DNA extracted by kit was higher, but its quantity was lower compared with CTAB method. Although with higher yield of DNA, the CTAB method led to a high content of impurities, and DNA degradation was serious. The CTAB protocol was optimized by the following strategies: 1) Adjusting reagent dosage according to the amount of sample; 2) Rinsing samples by PBS with pH 8.0 to eliminate impurities; 3) The dosage of lysozyme was increased by 20%, the lysozyme reaction time was reduced to 0.5-1h; 4) Using SDS and proteinase to lyse bacteria and remove protein; 5) Using CTAB/NaCl to replace CTAB and shortening the reaction time to 10min; 6)Using chloroform/isoamyl alcohol(24: 1) and phenol/chloroform/isoamyl(25: 24: 1) to replace chloroform. Repeating extractions was conducted to remove proteoglycan. The efficiency of the traditional CTAB extraction protocol was improved after these optimizations.(3) The detection limit at CT 31 corresponded to 4.54±2.9(cfu/mg) JCM1149 in tilapia intestinal samples, while the limit at CT 32 corresponded to 25.6±18(cfu/mg) JCM1149 in zebrafish intestinal samples. Saturation and evacuation analysis showed that the number measured by qPCR was higher than that by plate counting(P < 0.01). This suggested that qPCR can detect dead bacteria that still have probiotic effect, indicating a higher accuracy of qPCR compared with plate count method.(4) Evacuation test result showed that JCM1149 was significantly reduced in the gut of tilapia and zebrafish after JCM1149 bath(P < 0.01). JCM1149 decreased by two orders of magnitude in the gut of zebrafish at 24 h, while it decreased by one and two orders of magnitude in tilapia at 12 h and 72 h, respectively. Saturation test with series of JCM1149 concentrations during bathing showed that the concentration of JCM1149 in tilapia intestine reached a relatively stable state when the bathing concentration was greater than 105 cfu/mL, with no further increase at higher bathing concentration. In contrast, the concentration of JCM1149 in zebrafish intestine constantly increased with the increase of bathing concentration from 7.8×104 cfu/mL to 5.9×107 cfu/mL(P < 0.01), with no saturation at the highest concentration in this study.