Establishment And Application Of Four Pathogenic Bacterias On PRDC By Complex PCR

Porcine Respiratory Disease Complex (PRDC) is a general term of important syndromes which give rise to the respiratory symptoms. In recent years, with the rapid development of large-scale farming, a swine disease characterized by respiratory symptoms has been arisen, causing great loss in swine production. Studies show that Mycoplasma hyopneumoniae (Mhp), Streptococcus suis (SS), Haemophilus parasuis (HPS) and Pasteurella multocida (Pm) are considered to be the common pathogens of PRDC. Since the prevalence of Mhp, SS, HPS and Pm are not very clear and the above four pathogens have similar clinical symptoms, it is very difficult to diagnose, prevent, and treat the PRDC quickly and accurately. For this reason, this study aims to establish the quadruple PCR method with synchronous detection on Mhp, SS, HPS and Pm on the basis of previous research, providing a technical means for the rapid clinical identification and epidemiological research. In this study, the following two aspects of work have been carried out.Firstly, the single PCR method for the detection of Mhp has been established, and the complex PCR method for synchronous detection on Mhp, SS, HPS and Pm has been optimized. The specific primers are designed and synthesized applying certain software according to P36gene sequence. In addition, the best reaction system and reaction conditions of single PCR method for the detection of Mhp have been determined through optimization. Then the single PCR method is evaluated through sequence analysis, sensitivity test, specificity test and repeatability test. Based on the single PCR, the same temperature for the detection of the four pathogens are found out firstly, and Taq DNA polymerase, Mg2+, dNTPs and primers are optimized applying orthogonal design. Then, the PCR reaction time and cycle number are researched to determine the best reaction system and reaction conditions.Afterwards, detection for clinically suspected PRDC swine, clinically suspected non-PRDC swine and clinically assumed healthy swine in Anhui area are carried out respectively and bacteria isolation is performed with the combined method of quadruple PCR method, single PCR and conventional bacteria isolating technology. The results show that27strains of bacteria are isolated in total, including18strains of SS,4strains of HPS and6strains of Pm with no Mhp isolated. There is no significant difference between the test results of the quadruple PCR method and the single PCR method (P>0.05) with a coincidence rate of91.0%. A situation of higher infection in HPS and SS exists in various pig farms of Anhui province. HPS and SS together bear higher positive rate of15.84%(35/221), while the positive rate of the above pathogens together is0%. The distribution of pathogens differs in regions. Different pathogens are detected in each feeding phase.In conclusion, the quadruple PCR method established in this study is able to identify four kinds of pathogen with favorable specificity, sensibility, stability and repeatability. It also has advantages of simple, quick and economical. The results of experimental study show that the quadruple PCR method can be used for the identification of the four pathogens in PRDC directly, providing an effective technical means for the epidemiological investigation of PRDC in Anhui area.