| Haemophilus parasuis disease is caused by Haemophilus parasuis (Haemophilus Parasuis, HPs) which is a common bacterial disease of pigs, the disease is widely prevalent in our country and has resulted in major economic loss to our pig industry, fast and accurate diagnosis this disease and control epidemiological laws are very important to guide disease prevention and control. For the deficiencies of the existing methods, this study establish a PCR method for detection of HPs, and use this method conducted HPs molecular epidemiological investigation for Sichuan area, mastered the recent popularity and distribution of HPs Sichuan area, some molecular genetic characteristics of evolution, laid the foundation for carrying out the monitoring of the disease prevention and control for the Sichuan area, popular risk analysis, vaccine development and HPs gene databases and other.1. Establishment of PCR based on OMPP2 gene for detecting Haemophilus ParasuisThis study reference Genbank published HPs outer membrane protein P2 gene and select its conservative area designed PCR generic detection primers for all serotypes HPs, by exploring the optimization of annealing temperature, primer concentration and other conditions, successful establishment of a rapid PCR detection method, the standard strain HS80 PCR product sequences were determined and compared, the results indicate that the sequence homology of 97% to 99%, proved that the PCR methods has the accuracy for target sequences cloned. Then 15 serotypes HPs standard strains and streptococcus, E. coli salmonella, erysipelas bacteria etc. seven kinds of reference strains were validated specificity and reproducibility of the method, the results showed strong specificity reproducibility of the method and could detect 15 serotypes HPs strains. The sensitivity results showed that detection limit is 173 CFU, the sensitivity is higher than other. Meanwhile, the results also show that the method can quickly and accurately detect clinically suspected HPs diseased material. In summary. PCR method established in this study have specificity, high sensitivity, reproducibility, and not only be used for identification of clinically suspected isolates and also can be used for molecular epidemiological survey of HPs2. molecular epidemiological survey of Haemophilus Parasuis in Sichuan ProvinceDuring June 2013 to December 2013, this study collection 131 suspected HPs disease materials from more than 20 large-scale pig farms in Sichuan province Chengdu, Mianyang, Leshan, Meishan, Ya’an, Guang’an, Nanchong, Deyang and other 17 cities and counties, using the established PCR method to detect all disease materials,26 positive disease materials were detected, the detection rate is 19.8%, the positive samples were detected in pigs after weaning and conservation phases, source covering 11 regions, indicating that HPs have been widely popular in many parts of Sichuan, the main hazards is weaning pigs and conservation phases, so the disease should be given high priority. Through cloning and sequencing espp2 gene for 13 HPs isolated strains from positive disease, and analysis molecular characteristic for 13 sequences, the results showed that 13 HPs isolates strains from Sichuan are divided into two genes groups, and the nucleotide and amino acid sequences have a high degree of homo logy. It is suggested that a little difference between in HPs strains from Sichuan areaand have high homology. In summary, HPs have been widely distributed and popular in the Sichuan region in pigs after weaning and conservation phases, In Sichuan area,the popular HPs strains has a high homology from angle of pathogen molecule, this study has important guiding significance in science prevention and control for Haemophilus parasuis disease of Sichuan area. |
PDF Full Text Request