| Newcastle Disease(ND)is an acute,severe,and highly contagious powltry disease caused by Newcastle Disease virus(ND),which is characteristic of injury of digestive tract,respiratory tract and central nervous cells and led to heavy lossed to the poultry industry worldwide.At present,the emergence of new Newcastle disease genotype and subgentypes,such as class I,the conventional ND detection technologies in China is facing a challenge.The mixed infection of ND strains is very common in clinic,which seriously hinder the rapid diagnosis of virulent NDV and also the prevention and control of this disease.In this study,we have developed universal and virulent-stain-specific detection methods for NDV isolates,including conventional RT-PCR,fluorescent quantitative PCR with SYBR Green dye and TaqMan probe methods.The the specificity,sensitivity and feasibility of clinical application of these methods were detected.First of all,RT-PCR technology is the most conventional detection method with lowest cost in molecular biology.In this study,RT-PCR method was established with primer set AND-2 specific for conservative NP gene.The results show that all NDV strains of genotypes was detectible with our method,and the reactivity,specificity and sensitivity of which are no less than the RT-qPCR method recommended by the United States Department of agriculture.At the same time,three primer sets specific for virulent NDV were designed based on the motif F protein cleavage site of the virulent NDV strains,and RT-PCR methods were established and optimized.The results showed that primer set AND-5 identifed all of the NDV virulent strains,and had no response to the avirulent strains.Its reactivity,specificity and sensitivity were great.Then,fluorescent quantitative PCR with SYBR Green dye was established and optimized on the basis of the principle of RT-PCR detection method.Seven sets of NDV were designed.The results show that primer set AND-2 can detect all genotypes of NDV strains,and its reactivity,specificity and sensitivity are no less than the RT-qPCR method recommended by the United States Department of agriculture.At the same time,the primer set VID-5 can specifically identify genotype VI and VII virulent NDV;while primer set VID-7 can specifically identify genotype III,IV and IX.The RT-qPCR method established after optimizing the combination of primer VID-5 and VID-7 can specifically differentiate and diagnose NDV virulent strains.All the methods were good in reactivity,specificity and sensitivity.In order to further improve the specificity and sensitivity of the detection method,the experiment designed the fluorescence quantitative probe primers for the identification of virulent strains,and the probe primers QFS1-L,QFA and QFS2-S.The results showed that the detection method can identify all NDV strains,and it does not react with the avirulent NDV strains and all the other poultry diseases.Moreover,the sensitivity,reactivity and detection spectrum were significantly higher than the RT-qPCR probe method recommended by the USDA.Finally,the reactivity of established assays was detected with clinical samples.In this study,a laboratory animal detection model was established to infect chicks with NDV genotype VII virulent ZJ1 strain and genotype II vaccine strain La Sota.The laryngeal and cloacal samples were collected for detection.After comparing with the results of other detection methods,we found that the three detection methods established in this experiment are of good specificity,sensitivity and reactivity,and it can be used for clinical detection of NDV.Combined with NDV universal and virulent specific detection techniques,rapid virulence identification of NDV isolates can be achieved... |
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