| With the booming of poultry production, the incidence of avian disease of respiratory system had been rising and several pathogen infections were the main feature. Once mass infection broke out in poultry, it’s difficult to diagnose timely according to clinical signs and the optimal treatment time was delayed, which in turn, resulted that poultry production suffered huge economic losses. Avian influenza (AI), Newcastle disease (ND), Infectious Laryngotracheitis (ILT) and Infectious bronchitis (IB) were significant viral pathogens which could cause avian disease of respiratory system and was difficult to diagnose in clinical situations. So that it’s essential to establish a method which could rapidly detect multiple pathogen of poultry respiratory system disease simultaneously. As a high throughput detection technique, liquid chip was featured in convenience, accuracy and high throughput detection. In this study, the simplex and multiplex liquid chip detection methods for AIV (including H5, H7, H9subtype), NDV, IBV and ILTV had been preliminary established. The purpose of this study was to lay a foundation for the rapid and accurate detection of the avian respiratory system viral pathogen.HA gene sequences of AIV (including H5, H7, H9subtype), F gene sequences of NDV, N gene sequences of IBV and TK gene sequences of ILTV were found from GenBank respectively. Some biological software such as DNA MAN, DNA star, Primer5.0, Primer Express3.0were used for multiple sequence alignment analysis to screen conservative sequences. On the base, we designed specific primers and probes of these viral nucleic acid and coupled designed labeled probes with fluorescent coded microspheres. Next, asymmetric PCR/RT-PCR amplification products and probes-conjugated microspheres were hybridized. MFI values were analyzed according to the criteria with the Bio-Plex200system. Furthermore, the specificity, sensitivity and stability of the liquid chip system were determined and clinical samples were detected with the method.The singlex liquid chip methods for detection AIV (including H5, H7, H9subtype), NDV, IBV and ILTV firstly established on the optimization conditions, such as hybridization temperature and hybridization time. The results showed that the minimum detection concentration of AIV (including H5, H7, H9subtype), NDV, IBV and ILTV were 8.50×10ng/μL,2.87×10-4ng/μL,2.07X10-4ng/μL,2.61×10-4ng/μL,2.34×10-3ng/μL,2.05×10-3ng/μL respectively, which increased by10to100times compared with sensitivity of RT-PCR/PCR detection methods and the specificity and stability were also improve. Each chip only specifically detected corresponding virus nucleic acid which obtained a high MFI value and other viruses results were negative;50known samples were detected by these methods and the coincidence rate were100%. The results indicated that simplex liquid chip methods for AIV (including H5, H7, H9subtype), NDV, IBV and ILTV were more specific, sensitive and stable than roution method.On this basis, multiplex liquid chip method which could simultaneously detect AIV (in-eluding H5, H7, H9subtype), NDV, IBV and ILTV was established. Each virus probe on specifically detected corresponding virus nucleic acid which obtained a high MFI value, No cross-reaction was found in this method and reproducibility was well; The minimum detection concentration of each pathogen were5.44×10-4ng/μL,1.84×10-3ng/μL,2.65×10-4ng/μL,8.35×10-3ng/μL,1.50×10-3ng/μL,6.56×10-3ng/μL respectively.The sensitivity was increased by5to125times comparing to RT-PCR/PCR methods.50known samples were detected by this multiplex liquid chip method and the coincidence rate was100%.The singlex and multiplex liquid chip detection methods of AIV (including H5, H7, H9subtype), NDV, IBV and ILTV were successfully established in the study. Thus, a new technology platform for rapid, high-throughput detection of avian viral disease of the respiratory system was built and it also provided a new guideline for the detection of other viral pathogens. |
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