Cloning And Sequence Analyzing Of VBNC Campylobacter Jejuni's MRNA Suppressed Subtractive Hybridization Gene

Campylobacter jejuni is a kind of important gram-negative pathogenic bacteria.It can produce several bowel toxins, that will attack small and large intestine mucosal and cause acute colitis. According to statistics, the proportion lead to enteritis is three times the salmonella. It become the leading cause of food-borne diseases. While campylobacter jejuni come into the viable but non-culturable (VBNC) state, it can not cultured in normal Columbia Blood Agar medium. It produces toxins significantly decreased, but still has the ability to produce toxins. And this ability will recover under appropriate conditions. It is widely exist in livestock and poultry intestine, drinking water. Once enter the VBNC state, it will escape from detection. That will cause potential hazards to food safety.This research will use water as induction environment, temperature as the induction conditions. And the state and form of VBNC state was detected by acridine orange(AODC) and LIVE/DEAD kit staining, culturbility detected by plate count (PC). Results show that, the campylobacter jejuni in filtration sterilization water,37℃micro aerobic induction 48h, entered VBNC state. Bacteria changed from the normal form of the rods into spherical or approximate spherical, with the extension of time, all become spherical shape gradually. After the successfully induced, most bacteria still alive after kept 48 days in 4℃refrigerator. Save to 60 days, we found the bacteria gradually die through fluorescence staining observation test. Save to 100 days, we found most of the bacteria have died.Then we extract genes' mRNA through magneticaction. Using cDNA inhibitory cut hybrid method, and applied the DDRT-PCR, cloning, reverse Northern hybrid technology to deal with the normal and VBNC bacteria, adopt suppression subtractive hybridization method to clone and analysis the differential display of the two states(normal and VBNC). Results show that, these differential fragments are all belong to VBNC campylobacter jejuni through reverse hybrid and not to normal C. jejuni. The homology of these two fragments are higher than 98% compare to normal bacteria through Blast, the result is reliable.