Development And Application Of HRM For Detecting CVM

Complex vertebral malformation (CVM) is an autosomal recessive lethal genetic disease which was found in1999. A certain amount of carriers of CVM existed in our country because of the increasing number of importing holstein cows in recent years. It is important to establish a method which is rapid, simple, high sensitivity and specificity used in the export and import port.In this experiment, according to SLC35A3gene sequences published in GenBank, PCR primers were designed. The target gene was amplified by using the heterozygotic DNA as the template. After purification, the SLC35A3gene fragments were ligated to PMD18-T simple Vector, then were transformed into E. coli DH5a. Three standard plasmids including genotype G/G, G/T and T/T were constructed after screening and sequencing. Then two kinds of High Resolution Melting (HRM) curve anaysis were established on the basis of these standard plasmids. In HRM small amplicon genotyping method, specific primers are designed. The amplification products include the mutations site.By denaturization, we can find the difference of Tm value with the high and low temperature calibration in the amplification products. In this way, the genotype of the samples can be distinguished. In HRM with unlabeled probe genotyping method, unlabeled probe including mutations site was designed. To generate single stranded paired with probe, asymmetric PCR was performed. Then we distinguish the genotype of the samples through the Tm value of the probe amplicon. The two methods are absolutely right verified by directing DNA sequencing, and can be used in the detection of CVM. Eventually, a total of150Chinese Holstein bovine were assayed, and5CVM-carriers were identified, others are not CVM-carriers, verified by sequencing, the result is completely consistent.HRM analysis is a cheap, efficient and convenient method for genotyping, which was widely applied in many fields. In this study, HRM method for detection CVM is high throughput, cheap, simple and fast. It can be applied in export and import detecion.