Establishment And Preliminary Application Of PCR For Detection Of Helicobacter Pylori In The Milk And Faeces Of Dairy Cows

Helicobacter pylori (H.pylori, Hp) is responsible for upper gastrointestinal tract diseases and several extragastric diseases. However, the reservoirs and the pathways of transmission of Hp to humans have remained undefined. The studies show that Hp may be a kind of foodborne pathogenic bacteria, and cow milk and goat milk are the most likely sources of infection. At present, because of high sensitivity and the ability of detecting tiny amounts of Hp in the sample, polymerase chain reaction(PCR) related technologies are widely applied in the detection of Hp in clinical samples among many diagnosis methods of helicobacter pylori infection. This research aims to establish a PCR method for the detection of Hp in the milk and faeces of dairy cows by designing the specific primers corresponding to 16S rRNA、UreA and glmM gene, and apply it to detecting the Hp contamination in clinical samples successfully.1. The establishment of the PCR approach for the detection of Hp:The specific primers for detecting 16S rRNA、UreA and glmM gene from Hp were designed according to the published gene sequences in the GenBank database at the National Center of Biotechnology Information (NCBI). DNA from bacterial solution of Hp SS1(1×108 CFU/ML) was extracted as the PCR template DNA. Three target bands 777bp、411bp and 294bp can be specifically amplified by all the three pairs of primers respectively. The optimal PCR reaction system and conditions including template concentration, annealing temperature, concentration of primers, and cycle numbers are listed as follows:annealing temperatures of three target genes were 58℃～60℃,48℃～50℃ and 58℃～60℃ respectively. PCR reactions were performed in a final volume of 25μL containing 2μL genomic DNA as template,2 unit Taq PCR mix, and 0.4μL～0.6μL each primer (10μM) amplifying for 32～35 cycles. Under this condition, the sensitivity of PCR for 16S rRNA、 UreA and glmM gene from Hp was 101 CFU/ML、103 CFU/ML、103 CFU/ML. Compared with the sample of milk and faeces which infected by Hp articially, the sensibility of using UreA and glmM gene to detect Hp of the infected articial faeces was down to 104 CFU/ML. We cocluded that this PCR method was quick, specific, sensitive and efficient, which laid a solid foundation for identification of Hp infection in dairy cows.2. The application of the PCR method for the detection of Hp:In the present study, there were different degrees of Hp infection in the milk and faeces of 41 dairy cows which came from large-scale farms and free-range farmers. The detection rate of Hp in cow milk by PCR for 16SrRNA、UreA and glmM gene were 58.34%、34.15% and 14.63% respectively. While the positive rate of Hp in cow faeces were 19.51%,12.20% and 0%. In this sense, the infection rate of Hp in the milk and faeces of dairy cows by PCR for 16S rRNA and UreA gene was obviously higher than glmM.