Establishment And Application Of Nest-PCR And FQ-PCR Assay For Detection Of Haemophilus Parasuis

Haemophilus parasuis (HPS) belonged to pasteurella secco bloodthirsty coli.which caused mμLtiple serositis, arthritis and meningitis of pigs and bring economicloss to swine industry.Based on HPS16S rRNA gene，We successfully developedNest-PCR and fluorescence quantitative PCR methods and preliminarily applied inclinica detection.According to the specific conserved sequence of16S rRNA gene, we designedtwo pairs of specific primers for HPS Nest-PCR detection. HPS DNA as a template,primer concentration, dNTPs and the annealing temperature were optimized. Thespecificity, reproducibility and sensitivity of the test, detection in clinical samples ofsuspected infection of Haemophilus parasuis were performed.Based on the specificconserved sequence of16S rRNA gene, a pair of specific primers and a MGB probefor HPS Nest-PCR detection were designed.We establish the Nest-PCR and optimiedprimer concentration, probe concentration and annealing temperature.We conductedFQ-PCRwith quantitative10-fold serial dilutions of the recombinant plasmid andgenerated standard curve.The specificity, sensitivity and reproducibility of the testwere conducted and suspected of Haemophilus parasuis disease clinical samples weretested.The results indicated the Nest-PCR assay was succeefully established.Thespecificity of the Nest-PCR was tested,the results showed no products wereamplified,the fluorescence quantitative PCR assay was able to detect HPS and had nocross-reaction with other bacteria from swine.The sensibility of this assay ataained10copies of plasmid DNA,which was10times higher than routine PCR.The repetiton testindicated that the Nest-PCRwas repeatable.Clinical detection showed development ofthe method achieved the rapid diagnosis of HPS. The results indicated the FQ-PCR assay as well as a quantitative standard carve with good linearity(R~2=0.997)weresuccessfully established.The developed FQ-PCRassay with sensitivity up to1.98×10~1copy/μL,and its sensitivity of which more than of the toutine PCR.The specificityassay exhibited that positive signals could be obtained from recombinant HPS,but notfrom the genomic DNA of the other6kinds of pathogenic microorganismcating,Repetition of the test and between coefficients of variation were less than2.5%,which indicated that the repetition test indicated that the FQ-PCR wasreproducible.seventy precent positive results from thirtiech clinic suspicious HPSinfected sample were obtained,which showed the better sensitivity than that of thedetection results of the same thirtiech suspected samples by routine PCR15clinicallysuspected samples of Haemophilus parasuis infection diseased porcine tissues weretested, the resμLts showed that11were HPS positive, with100%consistency withNest-PCR diagnostic methods.