Constructing PRV-PPV-JEV Detection Microarray And Its Detection Technology

PRV, PPV and JEV detection microarray was constructed firstly in this study. Its detection technology was established which can simultaneously detect PRV, PPV and JEV, and can discriminate PRV gene deletion strains from field strains and low virulent strains. Major contents are as followed:1 Cloning and identification of PRV-PPV-JEV detection microarray target genesAnalyzing the molecular biological characters of PRV, PPV and JEV, primers of PRV, PPV and JEV were designed according to sequences from literatures and GenBank by ArrayDesigner software ver. 2. 0 . 16 targets were amplified by PCR or RT-PCR and cloned into PMD18-T Vector. 16 recombinant plasmids, assigned T/gB, T/gG, T/TK, T/gE, T/B1 NS1, T/Bl VP2, T/SR-1 NS1, T/SR-1 NS2, T/SR-1 NS3, T/SR-1 VP1, T/SR-1 VP2, T/C, T/PrM/M, T/E, T/NS1, T/NS5,were acquired and identified by PCR and sequencing. Targets gB , gG, TK, and gE amplified from PRV Fa strain, were fragments of gB, gG, TK and gE gene, with size of 324bp, 261bp, 177bp, 148bp,respectively. Targets B1 NS1, B1 VP2, from PPV Bl strain, targets SR-1 NS1, SR-1 NS2, SR-1 NS3, SR-1 VP1 and SR-1 VP2, from PPV SR-1 strain, were fragments of NS1,VP2, NS1, NS2, NS3,VP1 and VP2 gene, with size of 127bp, 471bp, 208bp, 550bp, 389bp, 209bp and 471bp, respectively. Targets C, PrM/M, E, NS1, NS5, from JEV SA14-14-2 vaccine strain, were segments of JEV C, PrM/M, E, NS1 and NS5 gene , with size of 238bp, 451bp, 439bp, 476bp and 448bp, respectively. Their nucleotide identities were above 98%. The cloned target genes were reliable for constructing PRV-PPV-JEV detection microarray.2 Constructing PRV-PPV-JEV detection microarrayTargets and probes were prepared for constructing PRV-PPV-JEV detection microarray in this study. The target genes, target gene spotting concentration, hybridization temperature, pre-hybridization time and hybridization time were determined. The quality of the constructed PRV-PPV-JEV detection microarray was evaluated. PRV-PPV-JEV Detection Microarray was successfully constructed on the base of above-mentioned terms.1. Preparation of the target genes Target genes were amplified by PCRwith template DNAs extracted from 16 recombinant plasmids. The location controller gene, 500bp was amplified by PCR with ADNA. PCR products were purified by isopropanol precipitation. Their concentrations was 300-900 ng/ u L.2. Preparation of probe Probes were labeled with fluorescein Cy3 by PCR. The final concentration of Cy3~dCTP in PCR was 2. 5u mol/L. The concentration of purified probes was 121- 748 ng/nL.3. The optimal concentration of targets was 200 ng/uL. Microarray system sensitivity was 3pg/u L probe. Pre-hybridization and hybridization time were determined to be lh and 6h, respectively. Specific target genes and hybridization temperature were selected out according to hybridization result at 44-60°C.4. Data analysis and quantification were based on patterns of Total Signal Intensity and Signal Intensity Median with QuantArray? software Ver. 3.0. Signal-to-noise ratio (SNR) was total signal intensity of sample to background value.5. Fabrication of microarray and its quality control PRV gB, PRV gG, PRV TK,PRV gE, PPV Bl VP2,PPV SR-1 NS2, PPV SR-1 NS3, PPV SR-1 VP1, JEV C, JEV PrM/M, JEV E, JEV NS1 and JEV NS5 were determined to construct detection microarray. Target genes and location controller gene were diluted to 200ng/ u L with Baio? spotting buffer, distributed onto the surface of aminated glass slide with SpotArray?24 Microarray Printing System. The spotting parameters were spot diameter 220urn and center-to-center 650urn. Microarray dried at room temperature overnight, hydrated at 60-80 "C for 10s, UV cross-link for 25min, washed in 0.2%SDS for 5min, and dried. PRV-PPV-JEV Detection Microarray was considered to be qualified according to SNR^l. 5 or sample median signal intensity =^1000, low diversity among the same batch of microarrays.3 Detection technology of PRV-PPV-JEV detection microarray Preparation method of Sample probe was established. Probes were labeled with fluorescein Cy3 by multiplex PCR with mixed primers HY, such as HY1(PRV gB, PPV Bl VP2 and JEV C),HY2(PRV gB, PPV SR-1 NS2 and JEV PRM/M), HY3(PRV TK, PPV SR-1 VP1 and JEV E), HY4(PRV TK, PPV SR-1 NS3, JEV NS1 and JEVNS5) and HY5(PRV gG, PRV TK and PRV gE).The PRV-PPV-JEV detection microarray was verified to be specific, reproducible and could be stored at room temperature for at least 4 months.Its hybridization positive standard was SNR ^1.5 or sample median signal intensity^lOOO or clear microarray scanogram.Eight strains of PRV, five of PPV and JEV SA 14-14-2 were detected with PRV-PPV-JEV detection microarray. The result showed that PRV-PPV-JEV detection microarray could detect PRV, PPV and JEV simultaneously and discriminate gene deletion strains from PRV wildtype strains and low virulent strains.