Study Of The Technique Of Simultaneous Detection Of Porcine Parvovirus And Haemophilus Parasuis By Gene Microarray

Porcine parvovirus and Haemophilus parasuis are two kinds of pathogens which are seriously harmful to the pig industry.The gene chip detection technology has the advantages of high flux,parallelism and automation.This study intends to establish a gene for simultaneous detection of these two pathogens Chip detection technology for the diagnosis of clinical swine disease to provide fast,high-throughput detection technology.Two pairs of asymmetric PCR primers were designed using Primer Premier 5.0software according to the sequence of porcine parvovirus(PPV)VP2 protein gene and the inf B gene sequence of Haemophilus parasuis(Hps).PCR and PCR were used to test the specificity of the four viruses,such as pseudorabies virus,porcine circovirus type II,porcine respiratory and reproductive disorder syndrome virus and swine fever virus.Streptococcus suis type 2,Staphylococcus aureus,Salmonella and The results showed that the two pairs of primers were highly specific,only the porcine parvovirus and Haemophilus parasuis were positive,and the other pathogens were negative.The results showed that the two pairs of primers were highly specific.Three replicates were performed under the best conditions of PCR amplification to verify the reproducibility of the two pairs of primers.Two specific probes were designed at the gene sequence of the primers,and the 5 'end of the primer was labeled with Cy3 at the 5' end of the primer.The primer was synthesized and diluted with TE buffer to 10?M/L.Into 5?M/L,10?M/L,15?M/L,20?M/L four different concentrations of spare.The PPV and Hps gene microarray were prepared by using the probe with the best concentration.The PPV and Hps were used to detect the gene chip and fixed with a wet box with fixed synergist.The PCR products were amplified by asymmetric PCR,and they were denatured and hybridized respectively under different conditions.After the hybridization was completed,the chips were washed in the preheated washing solution 1,the washing liquid 2 and the washing liquid 3 were washed for 2 min;after centrifugation,the chip was scanned with a gene chip scanner to observe and save the obtained picture.In the hybridization process,PPV was used as an example to optimize the target probelength,hybridization humidity and temperature.The results showed that the growth of the target probe and the increase of the hybridization humidity were beneficial to enhance the hybridization signal during the hybridization process.The hybridization signal of PPV gene chip was the best at 47 ?,and the hybridization signal of Hps gene chip was the best at49 ?.The results showed that there was no significant difference in the fluorescence signal between different concentrations of PPV and Hps gene,and the hybridization signal was 1h higher than that of hybridization for 2h.Based on the above hybridization results,5?M / L concentration of PPV and Hps probe were used to prepare the simultaneous detection gene chip.The results showed that PPV and Hps were successfully detected in the case of hybridization at 48 ? for 1 h,and the fluorescence signal was clear.Finally,the microbes were hybridized three times under the same conditions,and the chips of different batches were hybridized three times under the same conditions.The results showed that the chip was reproducible.The immobilized chip was placed at 4 ? for 2months.Under the same conditions,the scanning results showed that the fluorescence signal was good,indicating that the chip had good stability.In conclusion,the PPV and Hps gene chip detection technology was established in this study to achieve the purpose of detecting pathogen.Which laid a foundation for the prevention and eradication of animal diseases.It also provided theoretical basis for the diagnosis of gene chip and the study of animal epidemic situation.