Newcastle Disease Detection By Gene Chip And Typing Of Newcastle Disease Virus By RT-PCR Test Coupled With Restriction Endonuclease Analysis

Newcastle disease(ND) is an acute, highly contiguous avian infectious disease brought by Newcastle disease virus(NDV). Newcastle disease is A violent contagion classified by OIE. Newcastle disease virus is a member of Rubulavirus,Paramyxovirinae,Paramyxoviridae, and a negative RNA virus which has vesica membrane. It exists in all tissues,apparatus, body fluid,secretion and excrement. For the time being,we use traditional pathogen separation identification and serology methods such as HI, compared with molecular biology methods of USA and Japan, and have a gap with them. After entering into WTO,with the development of poultry products trade, the gap of disease detect technique will be stood out, so we have lower international competition. To change the disadvantage,it is urgent to research the advanced disease detect methods.OIE recommend NDV virulence test are inoculation NDV and molecular biology in vitro,inoculation NDV includes ICPI,IVPI and MDT test,but these need long time and maybe scatter the virus. For the time being,the NDV detection of CIQ includes cloaca tampon separate virus,ELISA,HI and RT-PCR is trying now. In recent years,some scholars abroad make use of molecular biology such as RT-PCR to sequence DNA and nested-PCR to research the detection of NDV. All these molecular biology in vitro have the characters of rapid,nicety,no infection,etc.,recommended to NDV virulent test by OIE. Some western countries and Japan have use these methods to quarantine NDV,it provide broad foreground for the development of NDV detection. Gene chip develop very quickly in molecular biology and disease detection in recent years. We can make use of chip to analysis virus gene group; construct mutation detection technique of heredity diseases and become normal detection technique of many heredity diseases; supervise the changes of cell gene expression,research pathogenic theory of virus; differentiate bacteria gene types and identify bacteria strains,provide base data for screening and supervise drug-fast gene. For the time being,the gene chip is used to disease inspection in research of entry and exit inspection and quarantine. All genome chip for inspecting SARS was prepared by Animal and Plant Inspection Institute of AQSIQ. All these improve the inspection technology. We have not found the report concerning NDV gene chip. 1. Gene chip detect NDV We design 30 of 20 mer oligonucleic acid fragments to detect NDV based on the oligonucleic acid design principle. The results of 6 strains detection show that probe sites detect the positive signals of 6 samples and negative and blank have no signals to confirm the stability and sensitivity of the chip. The results of AIV,ILTV,IBV,IBDV detection show that the hybrid signals are accurate and specified. The chip is sensitive,accurate,specified, stable,simple and cheap,so it provide a new way for NDV detection,suitable for screening and rapid test,has important meaning to the entry and exit animal inspection and quarantine. 2. RT-PCR coupled with restriction endonuclease analysis differentiate NDV strainsThe amply sequence is 4893～5031 of F gene of NDV,has a BglⅠsite in avirulent but not in virulent strains. We use RT-PCR coupled with restriction endonuclease analysis to type 45 NDV strains,and the results are unanimous with MDT of OIE standards. 41 strains only have 202bp segment,are virulent,the same as MDT results. The rest 4 strains (No. 8,9,10,44) MDT are over 60h,2 strains (No. 10,44) only have 202bp segment,are avirulent, and 2 strains ((No. 8,9) have 135bp and 67bp besides 202bp,indicate that these 2 strains have virulent and avirulent sequence,suspect infected by virulent; LaSota digest result indicate only have avirulent sequence. Two vaccines in China results are avirulent sequence. All these results show that the method can differentiate virulent and avirulent,vaccine.Amplifying 45 isolate strains by RT-PCR,the result further confirm other NDV isolate serology results(HA,HI,etc),coupled with...