Detection Technology Research Of Simultaneous Detection PRV-PPV-PCV2 Visual Microarray

With the development of China’s pig industry in the direction of intensive and scale, a variety of disease-prone and pathogenic mixed infection is widespread that has caused a certain degree of loss to the pig industry. Porcine Pseudorabies Virus, Porcine Parvovirus and Porcine Circovirus Type 2 are the 3 important disease pathogens result in porcine reproductive disorders. These 3 diseases clinical signs are similar, need to be in the differential diagnosis. This study aimed to carry out the construction of simultaneous detection PRV-PPV-PCV2 microarray visualization and the establishment of its testing methods and preliminary evaluation of the application. The study successfully prepared PRV-PPV-PCV2 simultaneous detection PRV-PPV-PCV2 and established its detection methods. The test results do not need a scanner to detect and the naked eye can see. The main research is as follows:1. Construction of probe gene recombinant plasmid bacteriaTwo detection probe gene recombinant plasmid bacteria of T/NS1 and T/ORF22 and Two positioning probe gene recombinant plasmid bacteria of T/PUC18 and T/GAPDH used in the preparation of PRV-PPV-PCV2 microarray visualization was successfully constructed in this experiment.According to the gene sequences in Genbank included, each one pairs of specific primers of PPV, PCV2, PUC18 and GAPDH were designed with software. Genomic DNA were extracted from PPV inactivated vaccine, PCV2 laboratory isolates with DNA extraction kit. PCV2 ORF2 gene, PUC18 gene and GAPDH gene were amplified with PCR, using the genomic DNA as template. The length size is 100 bp.122 bp,217 bp and 257 bp respectively.4 probe gene recombinant plasmid bacteria were constructed by connected the 4 genes to pMD19-T carrier and then conversed to E. coli DH5α respectively. Probes recombinant plasmid bacteria were successfully screened out with PCR amplification and nucleic acid sequence determination, then named as T/PPV-NS1, T/PCV2-ORF2, T/PUC18 and T/GAPDH. The long-term preservation of probe gene was established with the construction of Recombinant plasmid bacteria. It is one of the core technology of microarray preparation.1. Construction and optimization of detection technologyThe preparation of microarray visualization and its optimization of testing procedures were studied in this experiment. And the microarray quality and outcome criteria were identified. PRV、PPV、PCV2 can be detected with microarray prepared, and PRV vaccine strains and wild strains infection can be distinguished.Construction of microarray visualizationTake probe gene recombinant plasmid as template, amplified by PCR, purified by ethanol precipitation to prepare probe gene. Then probe genes of a certain concentration were diluted with Baio(?)spotting buffer. After denatured spraying sample, fixed in oven for some time, then the chip was successfully prepared.Labeling target genesExtracted the viral genomic DNA as template, then amplified and labeled target genes by PCR. The upstream primer that 5’-end biotin-labeled was added to the PCR amplification marker system, labeling with PCR amplification.Optimization of microarray preparation and testing technologyIn this test, the concentration of the spray sample probe gene, repeat spray sample times, probe gene fixed time, hybridization time and chromogenic substrate time were optimized, and microarray testing procedures were determined:the concentration of probe gene was 100 ng/ul. The chip was successfully prepared after spraying samples one time with instrument and placed in oven 2 h. The chip is placed at 44℃ prehybridization 1.5 h; Target genes were denatured in PCR instrument 95℃10 min, immediately cooled on ice 5 min. Take 100 ul of target gene hybridization 1.5 h. Then the chip is washed successively with lotion Ⅰ, Ⅱ,Ⅲ 5 min; Closed 37℃ 40 min; Again washed by the above conditions after 37℃ enzyme-linked 30 min; Chromogenic substrate 8 min, washed with distilled water. Then hybridization is completed.Determination of microarray quality and outcome criteriaThe quality of the chip and the criteria for the results were ensured:The quality of microarray based on positive control generated brown spots, blank no spots generated, and negative control generated no spots or a lighter spots. According to the depth of the spot color of target genes, negative control and blank control contrast to determine whether the test result is positive.1. Evaluation of detection technology of microarray visualizationSpecificity, sensitivity and shelf life that determine the quality of the chip was studied in this experiment. The accreditation chips were applied to the detection of clinical samples, comparing the result with PCR detection.Specificity, sensitivity, and shelf life testingSpecificity, sensitivity and shelf of the constructed microarray were studied. Chips were hybridized with PRV, PPV, PCV2, CSFV, PRRSV, JEV PCR products with biotin-labeled to test their specificity; Each probe gene plasmid DNA, PCR amplification, respectively for 10 x series times diluted then hybridized with chip to detect the sensitivity; PCV2 detection gene chips, for example, the saved 30 days,60 days,90 days and 120 days chip was extracted to hybridize for the shelf life testing. The results showed that:Chips hybridized with CSFV, PRRSV, JEV were negative and no cross-reactivity between the PRV, PPV, PCV2 each target gene. Showed that the established chip good specificity; The sensitivity of PRV-gB, PRV-gE, PPV-NS1 and CV2-ORF2 probe genes were 18.3 pg/μl, 17.4 pg/ul,11.7 pg/ul and 13.2 pg/ul respectively, and they are all 10 times higher than PCR.; the chip could still be effectively detected after saved for 120 days.Detection of clinical specimensThe chips were applied to the 103 clinical samples detection of Sichuan, Chongqing, Fujian, Guizhou, Gansu. Genome DNA was extracted from the dead and sick. Target genes were hybridization detection with chip after PCR labeled, then compare the alignment with PCR result. The result showed that PRV, PPV, PCV2 positive rate was respectively 22.3%(including vaccine strains positive rate of 2.9%, the positive rate was 19.4% in wild strains),8.7%,78.6%. PRV and PPV mixed infection rate was 8.7%, mixed infection with PCV2 and PRV was 22.3%, PPV mixed with PCV2 infection was 8.7%, mixed infection rate of 8.7% for the three diseases. The results showed that mixed infection PRV, PPV, PCV2 clinically were very common. Chip technology and the technique of PCR detection total coincidence rate was above 97%, indicating the quality of built chips were reliable and could be used for the detection of clinical samples.