| Avian leucosis virus（ALV）,Marerk’s disease virus（MDV）,Infectious butsal disease virus（IBDV）are the most common agents that cause poultry immunosuppressive disease.Newcastle disease virus（NDV）,Avian influenza virus（AIV）,Infectious laryngotracheitis virus（ILTV）are the most common agents that cause poultry respiratory diseases.It is difficult to carry on multi-pathogen and multi-pathogen types immediately detection,and no high-throughput co-detection methods were available for these 6-pathogen,and any pathogen types as well.which significantly hindered the quickly clinical identification of avian diseases.Inthepresentstudy,visualmicroarraysforimmediatelydetecting ALV-MDV-IBDV-NDV-AIV-ILTV and H5-H7-H9-N1-N2-N9 were developed combined with gold label silver stain（GLSS）.The main results as follows:1.Development,parameter optimization,and evaluation of a visual microarray for immediately detecting ALV-MDV-IBDV-NDV-AIV-ILTV1)Development and parameter optimization of a visual microarray for immediately detecting ALV-MDV-IBDV-NDV-AIV-ILTV.The sequences of ALV-ENV,MDV-MEQ and IBDV-VP2 gene were cloned from the genomic sequences of ALV,MDV and IBDV.After probesdesign,avisualmicroarrayforimmediatelydetecting ALV-MDV-IBDV-NDV-AIV-ILTV was developed（MicroarrayⅠ）.The optimized concentration of probes were 50μmol/L,and the optimized temperature and time for hybridization were 40℃and 2 h,respectively.The optimized concentration of nanogold-streptavidin was 4μg/mL with the staining time was 5 min.As a result,the silver staining was observed obviously.2)Quality evalution of the MicroarrayⅠ.For evaluating the quality of the microarray developed previous,the results of specificity evaluation showed that no cross binding was observed among ALV,MDV,IBDV,NDV,AIV and ILTV,with the avian infectious bronchitis virus（IBV）as negative control for MicroarrayⅠidentification.The optimized sensitivity of the microarray was 1 pg/μl.The microarray was stored at least 60 d at room temperature and75 d at 4℃.2.Development,parameter optimization,and evaluation of a visual microarray for immediately detecting H5-H7-H9-N1-N9-N21)Development and parameter optimization of a visual microarray for immediately detecting H5-H7-H9-N1-N9-N2.The sequences of H5-HA,H7-HA,H9-HA,N1-NA,N9-NA,N2-NA were cloned from the genomic sequences of H5N6,H7N9,H9N2 and the conserved sequence of H5N1-NA.A visual microarray for immediately detecting H5-H7-H9-N1-N9-N2was then developed（MicroarrayⅡ）.The optimized concentration of the probes were 50μmol/L,and the optimized temperature and time for hybridization were 45℃and 2 h,respectively.The optimized concentration of nanogold-streptavidin was 4μg/mL with the staining time was 7 min.2)Quality evalution of the MicroarrayⅡ.Silver staining was observed obviously.The results of specificity evaluation showed that no cross binding was observed.No cross interation of microarrayⅡwas observed with ALV,MDV,IBDV,NDV,IBV,ILTV,respectively.The minimize concentration for H5 and N1 microarrays was 1×10-10ng\ul,while1×10-8ng\ul and 1×10-7ng\ul for H7 and N9,respectively.The minimize concentration for H9and N2 microarray was 1×10-8ng\ul.The optimized sensitivity of microarrayⅡwas better than the PCR method.The microarray was stored at least 90 d at 4℃.3.Clinical application of MicroarrayⅠand MicroarrayⅡ.In this study,155 clinical samples presumed to be infected with ALV,MDV,IBDV,AIV,ILTV,NDV,were collected from Anyue,Pengzhou,Luzhou,Guangyuan,Zigong,Leshan,Guanghan,Wenjiang,Chongqing,Rongxian and Wannan area.The results showed that the positive detection rate of the 6 viruses（ALV,MDV,IBDV,NDV,AIV,ILTV）was 7%,1.2%,0.6%,5.8%,14.8%,0%,respectively.The mixed infection rate of ALV-AIV,NDV-AIV,ALV-MDV-IBDV,ALV-AIV-NDV was 4.5%,2.5%,0.6%,1.2%,respectively.The sensitivity of the microarray was showed better than that of RT-PCR/PCR,shared over 98%coincidence.The positive detection rate of the 6 avian influenza subtypes virus（H5,H7,H9,N1,N9,N2）was 4.3%,0%,43.4%,0%,34.7%,4.3%,respectively,in which the detection rate of H9N9 and H9N2 was 21.7%and 4.3%,respectively.The mixed infection rate of H5and H9 was 4.3%.The sensitivity of the microarray method was 100%,with the specificity was over 92.8%.The results of microarray shared 95.6%coincidence with that of the PCR/RT-PCR method,with the Kappa value was over 0.80.In conclusion,our study developed visual microarrays for immediately detecting ALV-MDV-IBDV-NDV-AIV-ILTV and H5-H7-H9-N1-N9-N2 with reliable specificity,sensitivity and stability,and was available for clinical diagnosis.These results will provide efficient tools for the diagnosis of avian diseases. |
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