Establishment Of Multiplex PCR For Detection Of Bacteria Responsible For Porcine Respiratory Disease

Streptococcal disease,Haemophilus parasuis disease and Actinobacillus pleuropneumoniae disease are the serious perplexs of three common infectious bacterial disease in the pig breeding.Three types of bacteria can cause a serial of respiratory symptoms.In which,Haemophilus is normal bacteria in the upper repiratory tract of swine.Streptococcus suis is a Human zoonotic diseases pathogens.The three pathogens mainly infect young piglets and nursery pigs and can cause a higher morbidity and mortality,especilly it was difficult to distinguish them by the clinical symptom so that they have caused a great ecomonic losses to the global pig-producing.The study construct four multiplex PCR detection methods that based on cps and gdh gene of streptococcus suis,Apx IV and cps gene of Actinobacillus pleuropneumoniae and 16S rRNA gene of Haemophilus parasuis.Through the clinical test,four methods had been proved to have the very good repeatability,sensitivity and specificity.The multiplex PCR method for SS,APP,HPS was established through optimizing the system and condition of the single PCR and the multiplex PCR reaction.200 nasal swabs of apparently health pig were detected by the multiplex PCR.Results:the multiplex PCR method we established could simultaneously amplify SS,APP and HPS in the same sample.Pasteurella Multocida,Escherichia coli,Actinobacillus suis,Staphylococcus aureus,Salmonella and swine Erysipelas were detected by the multiplex PCR respectively and no special amplification products were found in the results.The minimum concentration of detecting SS,APP and HPS were 104CFU/mL,103 CFU/mL,103 CFU/mL,respectively.The sensitivity of SS,APP and HPS were 104CFU/mL,104CFU/mL and 104CFU/mL,respectively.Among 200 pig specimens from Jiangsu province,the total positive rate was 93.5%(187/200)and within them,the positive rate for SS,APP and HPS was 80%(160/200),1.5%(3/200)and 22.5%(45/200)respectively.The total coincidence rate between the bacterial isolation and the multiplex PCR for 30 clinical disease samples was 93%(28/30).This multiplex PCR is sensitive,specific and rapid method for the detection and identification of major bacteria in porcine respiratory disease complex.The multiplex PCR method was quickly and effectively and the results would be obtained in 4.5 hours.The multiplex PCR method for SS serotype 1、2、7、9 could simultaneously amplify SS serotype 1、2、7、9 in the same sample.Pasteurella Multociday Actinobacillus pleuropneumoniae,Escherichia coli,Actinobacillus suis,Staphylococcus aureus,Salmonella and swine Erysipelas were detected by the multiplex PCR respectively and no special amplification products were found in the results.The minimum concentration of detecting SS serotype 1、2、7、9 all were 103CFU/mL.This multiplex PCR is sensitive,specific and rapid method for the detection and identification of major bacteria in porcine respiratory disease complex.The multiplex PCR method was quickly and effectively and the results would be obtained in 4.5 hours.The multiplex PCR method for APP and serotype 1、3、5、7 could simultaneously amplify APP and serotype 1、3、5、7 in the same sample.Escherichia coli,Pasteurella Multocida,Streptococcus suis,Erysipelas,Haemophilus parasuis and swine Salmonella were detected by the multiplex PCR respectively and no special amplification products were found in the results.The minimum concentration of detecting APP and serotype 1、3、5、7 all were 103CFU/mL.This multiplex PCR is sensitive,specific and rapid method for the detection and identification of major bacteria in porcine respiratory disease complex.The multiplex PCR method was quickly and effectively and the results would be obtained in 4.5 hours.The multiplex PCR method for APP serotype 2、6、8、12 could simultaneously amplify APP serotype 2、6、8、12 in the same sample.Pasteurella Multocida,Escherichia coli,Actinobacillus suis,Staphylococcus aureus,Salmonella and swine Erysipelas were detected by the multiplex PCR respectively and no special amplification products were found in the results.The minimum concentration of detecting APP serotype 2、6、8、12 all were 103CFU/mL.This multiplex PCR is sensitive,specific and rapid method for the detection and identification of major bacteria in porcine respiratory disease complex.The multiplex PCR method was quickly and effectively and the results would be obtained in 4.5 hours.