Establishment And Application Of Four Kindspathogens Of Swine PCR Diagnostic Method

Actinobacillus pleuropneumoniae is the major pathogen of Porcine contagious pleuropneumoniae. After the APP infection, Pigs will produce acute fibrinous pneumoniael, lung hemorrhagic necrosis even death, or evolve into chronic infection, although no obvious clinical symptoms, but slow growth even become stiff pigs. After the attack the pigs of carrying pathogens may become the infection source of the outbreak of the disease. In recent years, Actinobacillus pleuropneumoniae secondary infection and mixed infection with other pathogens become more and more serious and cause clinical symptoms becomed more complex and difficult to distinguish. So far, the APP could be classified into 15 kinds serotypes based on capsular polysaccharides. According to the training process whether need NAD, the APP could be divided into biotypes I and II, our country was mainly popular biotype I. Since the APP serotypes are numerous, the existing commercial vaccines do not cover all serotypes, in addition, making type-specific serum process was complex and time-consuming, which bringing the great difficulties to diagnose and prevent the disease. Therefore, the identification and somatoype of the APP as well as taking further according to the pathogen take corresponding vaccines vaccination and medication therapy are great significant to comprehensive preventing and controling the disease.In this study, taking advantaging of biotype I APP standard strains maked the 13 standard positive serums, after the gel diffusion test(GDT)measuring the titer of the standard positive serotype 5 was 1: 8; the titers of the standard positive serotype 2, serotype 4, serotype 9 and serotype 15 were 1:16; the titers of the standard positive serotype 1, serotype 3, serotype 6, serotype 7, serotype 8, serotype 10, serotype 11 and serotype 12 were 1:32.In recent years, due to rapid, simple features, the multiplex PCR method developed rapidly and used widely. According to the exotoxin apx I, apx II, apx III and apx IV of the APP designed various specific primers to research multiplex PCR molecular typing method, by a step multiplex PCR method could distinguish seven serotypes of the APP serotypes 1 to 13(type 1, type 3, type 4, type 5, type 8, type 10 and type 12).Porcine respiratory disease was the main reason of death of leading to the nursery pigs, growing pigs and growing and fattening pigs. Haemophilus parasuis, Swine streptococosis, Pasteurella multocida and Actinobacillus pleuropneumoniae were the main pathogens of causing Porcine respiratory disease complex(PRDC). In this study, against the 16 S r RNA gene of Haemophilus parasuis, the gdh gene of Swine streptococosis the kmt gene of Pasteurella multocida, and the apx IV gene of Actinobacillus pleuropneumoniae designed four pairs of specific primers which could amplify the specific fragment of 821 bp, 689 bp, 457 bp, 319 bp. Based on the individual PCR of APP, further, the several major factors of affecting the PCR(r Taq polymerase, Mg2+ concentration, d NTP concentration and primers concentrations)were optimized to establish a multiplex PCR detection methods. Specific test results showed that only Haemophilus parasuis, Swine streptococosis, Pasteurella multocida, Actinobacillus pleuropneumoniae could amplify specific bands, other results was negative, sensitivity test showed that Haemophilus parasuis lowest detectable amount was 112.5 pg/μL, Swine streptococosis lowest detectable amount was 11.7pg/μL, Pasteurella multocida lowest detectable amount was 8.7 ng/μL, Actinobacillus pleuropneumoniae lowest detectable amount was 267 pg/μL, by 9 repetitions multiplex PCR tests showed that the multiplex PCR had good reproducibility. Using this method, 579 clinical samples collected in the part of Hubei region were tested preliminary detection, there were 59 Haemophilus parasuis(10.2%),74 Swine streptococosis(12.8%),35 Pasteurella multocida(6%), 24 Actinobacillus pleuropneumoniae(4.1%), the multiplex PCR compared with the individual PCR results, the average positive rate was 95%. Showing that the method was practical and provided technical support for the pathogens of PRDC detection and disease prevention and controling.