Study On Applying Microarray System For Detection Of The Pathogenic Bacteria Of Cow Mastitis

Milk cow mastitis is one of these diseases which cause the biggest loss in cow industry. It is caused by many kinds of pathogenic bacteria, which involve a wide range of genera and species of bacteria, little existing methods can meet the requirement of quickness and parallelism. Here we present a new approach; based upon the epoch-making gene-chip technology, which is capable of simultaneously detecting and identifying the main pathogenic bacteria of milk cow mastitis. The genus or species specific probes on the microarray are targeted to the 16S rDNA, simultaneously involving the 23S rDNA. Micrroarray system was established. While primers amplified corresponding gene of bacteria, biotin was labelled. Amplified product hybridized with probes, according to the recorded signals, the sorts of pathogenic bacteria was determined.In this study, firstly, universal primers, universal probes and special probes located on the 16S rDNA conserved and variable region were designed for the detection of bacteria of milk cow mastitis, which were fit for the detection.Secondly, gene-chip detection technology based on the 16S rDNA which identified staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactisa, escherichia coli, pseudomonas aeruginosa and streptococcus uberis was built, we optimized the hybridization conditions. We also developed a set of instruments system to operate, which was simple, easy and cheap. According to the results of hybridization, we estimated the specificity, sensitivity and probability. Sensitivity of gene-chip detection technology is 10 times higher than that of PCR. staphylococcus aureus is 0.3cfu/ml, pseudomonas aeruginosa is 0.2cfu/ml, six sorts of pathogenic bacteria show a series of specific hybridization.Clinically, the sort of pathogenic bacteria was determined through the results of bacteria culture and judged by GB. But practically because of antibioticapplication there were clinical symptoms, but there was no results of bacteria culture. Gene-chip detection technology conquered the shortcomings of conventional detection. It was proved that the detection rate of six sorts of bacteria was higher than conventional detection, which was caused by antibiotic. Excluding this faction, we identified 122 milk cow mastitis samples, the results of both detection technology were consistent.A based detection system which employed 16S rDNA can identify six sorts of pathogenic bacteria rapidly, hybridization and data acquisition lasted about 6-7 hours, The sensitivity was improved greatly and specificity was high. Using gene chip to detect the pathogenic bacteria of cow mastitis is a sort of exploration, which is practical.