Development Of PRRS-CSF-PCVD Diagnostic DNA Microarray And Its Detection Method

Porcine reproductive and respiratory syndrome virus(PRRSV), classical swine fever virus(CSFV) and porcine circovirus type 2-(PCV2) are three important pathogens in swine diseases and caused enormous economical loss in swine production. We firstly developed PRRS-CSF-PCV2 Diagnostic DNA Microarray and its detection method. Major contents are three parts of the paper as followed. 1.Clone, identification and analysis of PRRS, CSF and PCVD targetsAccording to the sequences of PRRSV, CSFV and PCV reported in GenBank, conserved regions were found by Multiple Sequence Alignments (MSA).Targets were designed by ArrayDesigner 2.0 software based on their conserved regions.Fifteen targets were amplified by One-step RT-PCR or PCR,purified, ligated into plasmid pMD18-T, and transformed into E.Coli JM109.Fifteen positive recombinants were acquired and identified by PCR and sequencing,named as T/PRRS-PS9,T/U1b, T/E6, T/E8, T/C14P9, T/Y11, T/Y12, T/CSFPS5, T/CSF1, T/Npro, T/Erns, T/HCLVP7, T/PCVCQB7, T/PCV2P6 and T/PCVT3,respectively.Targets were analyzed by MSA of DNAMAN ver.5.2.2. The results were as followed.1) Targets PRRS-PS9(Accession No.AY775134), U1b(Accession No.AY775135), C14P9 (Accession No.AY775132), Y11 (Accession No.AY775133), Y12(Accession No. AY775136), amplified from C14-2 isolate, were fragments of 5'untranslated region (UTR), ORF1b,ORF5, ORF6, ORF7 of PRRSV North American type, 174bp, 301bp, 468bp,155bp and 176bp of length, respectively.E6 and E8,amplified from VP046 vaccine strain of Spain, were the segments of ORF6 and 5'-UTR of PRRSV European type, 350bp and 191bp of length, respectively. The nucleotide sequence identity of these targets with the same genotype strains reported in GenBank was above 90% and less than 70% with dissimilar genotype. Compared AY775132 with reference strain VR2332 and main attenuated North American type vaccine strains, their identities were above 99%, and with the majority of domestic isolates was about 85%. The results demonstrated that ORF5 exists great gene variation.2) Targets CSFPS5(Accession No.AY775139),CSFl(Accession No.AYY775140), Npro (Accession No. AY775138) and Erns(Accession No. AY775137), amplified from CSF sample, were fragments of CSFV 3'UTR. 5'UTR, N^pro and E^rns, 133bp,143bp, 293bp,301bp and 490bp of length,respectively.HCLVP7,ampIified from HCLV vaccine, was segment of E2 gene, 490bp of length. Comparing CSF targets with 1 to 6 genotype isolates of Pestivirus, their identities were CSFPS5,30-50%, 80-100%, 40-60%, 40-50%, 48%,48%;CSFl,60-70%,>95%,80%,64%.68.71%.68.54%;Npro,70-75%,85-100%, 75%,35-40%, 77%; Erns,55-60%,80-100%,30-35%,50-55%,55.94%,57.96%; HCLVP7, 60-65%,>90%,40%,60-65%, 64.51%, 66.55%,respectively.3) PCVCQB7, PCV2P6(Accession No.AY775142) targets, amplified form PCVCQB7 and P6 samples of PDNS, were fragments of PCV2 ORF2.PCVT3,from MS sample of PMWS, was fragment of PCV2 ORF1.Their lengths were 624bp, 566bp and 322bp, respectively. Comparing PCVCQB7, PCV2P6 with PCV isolates, their identities were above 90% with PCV type 2 isolates, and lower than 70% with PCV type 1 isolates.PCVT3 and PCV isolates, their identities were more than 80%. 2.The construction of PRRS, CSF and PCV DNA diognostic microarrayMicroarray preparation parameters, data analysis and quality control were studied. The. PRRS-CSF-PCVD Diagnostic DNA Microarray was successfully constructed.1) Targets were amplified by PCR from DNA extracted from recombinants and purified by isopropyl alcohol precipitation method. Orientating genes was prepared according to literature reported by Sanjie Cao(2004). Probes were labeled by PCR with fluorescein Cy3, optimized final concentration 2.5 u mol/L.The concentration of probe purified by DNA Fragment Purification Kit was 100.09-300 ng/u 1 . The targets and orientating gene were diluted to 200ng/ u 1 with BioF) 2 X microarray spotting buffer, distributed onto the surface of aminated glass slide by SpotArray?24 Microarray Printing System. The spotting parameters were spot diameter, 220 u m and center-to-center space,650 u m. The microarray was dried at room temperature for 6h,hydrated at 60°C water vapor for 10s,immobilized under ultraviolet for 30min,washed in 0.2% sodium dodecylsulphate(SDS)for 5min,and dried by centrifuge immediately.2) The hybridization dynamics of microarray showed that the optimal concentration were targets,200ng/ u L and probes,3-3000pg/ u L.As probe concentration exceeded 3000 pg/ y L, hybridization signal intensity tailed off due to energy transfer. The system sensitivity was 3pg/ U L.3) Data analysis and quantification were based on patterns of Total Signal Intensity and Signal Intensity Median with QuantArray? software Ver.3.0. Polluted spot was eliminated by replacing spot signal intensity ^ average ± 2SD with signal intensity median. Signal-to-noise ratio (SNR) was median signal intensity to median noise value ration based on Total Signal Intensity. As a general rule, the creditable signal intensity is ^ 1.5 times of SNR or ^ 1000 of median signal intensity (Signal Intensity Median).4) Targets hybridizing at 40-56°C showed that few targets had cross-hybridization due to fragment overlap and non-stringency hybridization condition,the other targets had high specificity. Targets PRRS_PS9,Y11,E6, E8. PCVCQB7, PCVT3,CSFl,Erns and HCLVP7 were used for constructing PRRS-CSF-PCVD Diagnostic Microarray .5) Quality of PRRS-CSF-PCVD Diagnostic Microarray was evaluated by hybridization test with perfect probes, reproducibility test. It is conclude that targets were reliable, mini-variance with different microarrays of the same batch.3 Application of PRRS-CSF-PCVD diagnostic DNA microarray1) Probes were labeled by multiplex PCR. After optimizing parameters of multiplex PCR, Primer mixture groups were HY1(PRRS_PS9,PCVCQB7,CSF1), HY2(E8, Erns , PCV2P6),HY3(Y1 l,PCVCQB7,HCLVP7),HY4(E6,Erns,PCV2P6),Two primer mixture groups were selected out. Probe labeling reaction system was determined. cDNA was synthesized by RT from RNA of PRRSV and CSFV. In 20 n L RT reaction system, the volume of RNA templet is 4.0 u L. Templet volume of cDNA are 10 u L in probe labeling PCR system with Cy3-dCTP.PCR Programme is Denaturing at 94 °C for 2 min; denaturing at 94"C for 30s,annealing at 55 °C for 30s,extending at 72"C for 30s,30 cycles; extending at 72 "C forlOmin,hold at 4°C.2) PRRS-CSF-PCVD Diagnostic DNA Microarray can simultaneously detect CSF,PRRS and PCVD, and distinguish PRRSV genotype. It has high specificity, high sensitivity and can be reused at least 10 times. It has no cross-hybridization with PRV,JEV,PPV,TGEV,A.PP,H.PS,E.coli and S.pp.3pg/ u L probe can be detected. The positive standard formulated as SNR ^ 1.5 or signal intensity ^1000.3) 20 clinical samples were detected by PRRS-CSF-PCVD Diagnostic DNA Micro-array. Positive sample percent were CSFV,15%(3/20); PCV2,5% (1/20); PRRS, 0(0/20). Co-infection ratios were PRRSV and CSFV,15%(3/20);PRRSV and PCV2, 35%(7/20); PRRSV,CSFV and PCV2,15%(3/20).The results indicate that we should take highly attention to co-infection of PRRSV,CSFV and PCV2.