Differentiation Of Four Poultry Diseases Using Asymmetric RT-PCR In Combination With Microarrays

Asymmetric reverse transcription polymerase chain reaction (RT-PCR) and microarrays were combined to distinguish four virus including Avian influenza virus (AIV), Newcastle disease virus (NDV), Infectious bronchitis virus (IBV), and Infectious bursal disease virus (IBDV) and hemagglutinin (HA) subtypes H5, H7, and H9, and neuraminidase (NA) subtypes N1 and N2 of AIV. The AIV matrix protein (M), and HA and NA genes, IBV nucleoprotein(NP) gene, NDV NP gene, and IBDV A fragment gene were cloned into plasmids. These genes were amplified from these positive recombinant plasmids which including the inserted target genes by polymerase chain reaction (PCR). The PCR products were purified and printed on the amino-modified slidesa as the probes. RNA was extracted from samples and labeled by asymmetric RT-PCR using a cyanine (Cy)3-labeled primers. The labeled complementary (c)DNAs were hybridized to the probes immobilized on the glass slidesa. After hybridization the microarrays were scanned, the hybridization pattern agreed perfectly with the known location of each probe, and no cross-hybridization could be detected. It is demonstrated that microarray based on asymmetric RT-PCR is an effective way to simultaneously distinguish AIV, IBV, NDV, and IBDV simultaneously:1. The preparation of the probesM gene of AIV, HA gene of H5,H7,H9 AIV subtypes and NA gene of N1,N2 AIV subtypes; NP gene of IBV; NP gene of NDV; segment A gene of IBDV and GAPDH gene(positive control)were amplified by PCR. The eighty-five PCR products were precipitated by ethanol,condensed to final concentration above 1μg/μl.2. Preparating and optimizating of the micoarrayAll the probes, negative and blank controls were printed on the amino-modified slides. In the process of sample preparation, RNA were extracted, RT-PCR were done regular forward primers and Cy3 labeled reverse primers. The amplification conditions were optimized. 3. Effectivity of microarrayTotal RNA was extracted from samples of AIV (subtypes H5N1, H7N1, H9N2), NDV, IBDV, and IBV preserved in the laboratory. The RNAs were labeled and hybridized with the microarrays. The results showed that the microarray has good specificity. Plasmids were diluted to different concentrations and subjected to RT-PCR and microarray, the results showed that the sensitivity of microarray higher than that of RT-PCR. The microarrays showed good reproducibility and a retention period of up to 60 days。4. Sample detection with microarray and comparison with other diagnostic methodCollected thirty cloacal swabs and tissue samples were tested by microarray, RT-PCR, and chicken embryo inoculation analysis. The accordance rates of the microarray detection with chicken embryo inoculation and RT-PCR were 100% (30/30) and 96.7% (29/30) respectively. 57 collected samples from field were subjected to RT-PCR and microarray. The results further revealed that the accordance rate of RT-PCR after microarray detection and chicken embryo proliferation was 96.4%.These results demonstrated that microarray combined with asymmetric RT-PCR is an effective way to simultaneously distinguish major subtypes of AIV, IBV, NDV, and IBDV simultaneously. Furthermore, the microarrays showed such characteristics as good reproducibility, rapid detection, accurate, high through-put, making it an effective new way suitable for large-scale epidemiological investigations and field screening of AIVs. Microarray in combination with asymmetric RT-PCR makes good foundation for further application.