Development Of A Multiplex PCR For The Identification Of Actinobacillus Pleuropneumoniae, Pasteurella Multocida And Hamophilus Parasuis As Well As A PCR-ELISA For The Identification Of Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae is a highly contagious pathogen responsible for hemorrhagic, suppurative and fibroid pneumonia. Pasteurella multocida is the causative agent for porcine pneumonic pasteurellosis. Hamophilus parasuis is a facultative pathogen which causes fibroid pneumonia, arthritis and meningitis of pigs. Actinobacillus pleuropneumoniae, Pasteurella multocida and Hamophilus parasuis are all pathogens commonly isolated from respiratory tract of pigs. They can cause sub-infections after the herds being infected with other diseases. Herds may also be vulnerable to other diseases after the infections. Clinical symptoms caused by the three bacteria are similar; multi-infection brings further difficulties for the diagnoses of the diseases. Based on this situation, a multiplex PCR was developed for the fast identification of the three bacteria.Non-specific amplification may occur when clinical samples are tested, especially when DNA from a variety of species are contained in the samples. If the non-specific amplicon is about the same size as that of the expected product, false positive results may be generated. In order to make qualitative analysis to PCR products and improve the specificity of the diagnostic assay, a PCR-ELISA test was developed for the identification of Actinobacillus pleuropneumoniae. 1. Development of a multiplex PCR for the identification of Actinobacillus pleuropneumoniae, Pasteurella multocida and Hamophilus parasuis.Primers for detecting Actinobacillus pleuropneumoniae (App-Apx-upper and App-Apx-lower) are designed according to the sequences of apxVIA of Actinobacillus pleuropneumoniae. Specific lower primer for detecting Pasteurella multocida and Hamophilus parasuis (Pm-16S-lower and Hps-16S-lower) are designed according to the species specific region of their 16S rRNA gene. Co-16S-upper and Co-16S-lower were yielded from conserved region of 16S rRNA gene. This set of primers can react with DNA templates from all the bacterial species included in this research and products generated are in the size of 1370-1400bp. Co-16S-upper is the common upper primer of Pasteurella multocida and Hamophilus parasuis. PCR products of Actinobacillus pleuropneumoniae, Pasteurella multocida and Hamophilus parasuis are in the size of 342bp, 485bp and 1258bp respectively. Expected positive results were generated when 12 serotypes of Actinobacillus pleuropneumoniae, 6 strains of Pasteurella multocida, 15 serotypes of Hamophilus parasuis as well as 25 isolates proved to be the up-mentioned three bacteria were tested. Negative results were generated when 15 other bacterium that could be isolated from pigs were tested. The lowest amounts of DNA detectable by the assay were 32pg, 34pg and 67pg as far as Actinobacillus pleuropneumoniae, Pasteurella multocida and Hamophilus parasuis are concerned. The PCR assay developed is suitable for the detection of Actinobacillus pleuropneumoniae, Pasteurella multocida and Hamophilus parasuis.2. Development of a PCR-ELISA for the identification of Actinobacillus pleuropneumoniaeThe objective of this research was to develop a diagnostic assay with higher specificity and sensitivity for the identification of Actinobacillus pleuropneumoniae. All serotypes of Actinobacillus pleuropneumoniae have apxVIA gene. Sequences of this gene are conserved especially in the region of 3'end and 5'end. A set of primer (Apx IV-upper and Apx IV-lower) were designed according to the conserved region of the apxIVA gene. 5'end of Apx IV-upper was labeled with biotin. In order to give a qualitative analysis of the PCR product, internal probes P1 and P2 were designed according to the sequences of the amplified region. P1 and P2 were of the same sequences; the only difference was that P2 was labeled with dig on the 5'end. The species specific probe detects PCR products with solution hybridization. The probe only anneals to perfectly matched DNA sequences when a comparatively high hybridization temperature is applied. This rule out the possibility of false positive results and improved the specificity of the assay. Signals were amplified twice in a PCR-ELISA system, namely PCR amplification and ELISA amplification. The sensitivity of the developed assay was 100 times higher than the sensitivity of simple PCR using the same set of primers. The unlabeled probe P2 was applied to give the assay a quality control. If DNA template of Actinobacillus pleuropneumoniae does exist in the system, the apxIVA gene is specifically amplified, then P1 will anneal to the amplicon specifically, P2 will interfere this hybridization and make OD value significantly lower. If the dropping of OD value also occurs in negative control samples it means the existence of non-specific hybridization, the result of the assay is questionable.