A New Real-time Quantitative PCR Detecting Method Of NDV And Application In Food Safety Control In Chicken

Newcastle disease is caused by Newcastle disease virus,mainly for chicken and turkey,acute,febrile,Septic,infectious disease.Because of the ubiquity of Newcastle disease,as well as the epidemic may occur throughout the year,it has become one of the most serious harm to the world poultry diseases.Therefore,Newcastle epidemiological monitoring,and make rapid and accurate diagnosis of its suspected cases,and thus control of the genetic variation of Newcastle disease,to protect the safety of consumers,the development of the poultry industry and reduce economic losses have very important.The traditional method of diagnosis for NDV virus is mainly depends on Isolation and identification and serological detection.(Mainly includes SPF neutralization tests,AGID,ELISA etc method).These methods always time-consuming,low sensitivity and highly dependent.For our country,according to the requirements of the national standard of NDV diagnosis,The main methods for the detection of NDV mainly includes:clinical diagnosis,HA,HI,ICPI,RT-PCR five kinds of detection methods.But the clinical diagnosis of suspected cases of infection by different and there are great individual differences,at the same time the diagnosis experience and habits of observation inspection personnel to the diagnosis result has great influence.Therefore,clinical diagnosis can only be used NDV preliminary diagnosis,HA,HI,ICPI methods have defects that complex operation,long experimental cycle,high positive rate.RT-PCR is a rapid and accurate detection method,but the products by agarose gel electrophoresis and EB staining,prone to contamination and false positive.With the rapid development of poultry breeding of the moment,we need to establish a rapid,accurate,and efficient detection method,and found that the control of food safety andconsumer protection,reduce economic losses.This study established establishment of the Taq Man fluorescent probe method on the basis of the traditional Taq Man one step QRT-PCR detection method based on RT-PCR to NDV.By using fluorescence quantitative PCR instrument to collect the fluorescence signal to realize the real-time monitoring of the entire PCR,the final results through the establishment of standard curve of unknown concentration oftemplate to implement quantitative analysis.Through this research proves that the method compared with the traditional method hashigher sensitivity,specificity and stronger stability.This study will also establish the detection method and the optimized is applied to the detection of clinical samples.Results by comparison with the traditional method,this method can be used to prove thedetection of clinical samples.This paper is divided into two parts to conduct the research:Optimization of conditions of one step QRT-PCR the of NDV detection.The NDV in chicken embryo method standard samples by SPF were cultured and used as template in the extraction,detection to detect the purity and integrity of q ualified.Through access to relevant literature,design specific primers and probe of NDV,On the basis of the reaction system identified by screening four optimization factor:Reverse transcriptase enzyme,dNTP,Mg2+,Taq.Using the orthogonal experiment to determine the optimal reaction conditions for each factorand interaction,and the establishment of one step QRT-PCR detection conditions for the detection of Newcastle disease virus.The kit of Real-time Quantitative PCR Detecting method for NDV research manufacture.At the first part the detection method and optimized the conditions of the foundation,developed a novel fluorescence monitoring of NDV QRT-PCR kit,The confirmatory test indicators such as specificity,sensitivity,reproducibility,stability,storage period of the kit assembly,obtain plentiful experimental data.In addition,according to the clinical sample detection requirements of the quality of the kitused in laboratory,the clinical data is important.These data demonstrate that the kit of NDV fluorescence quantitative RT-PCR detection reagent on the assembly has the advantages of rapid,high sensitivity,specificity,stability.It has great application value in monitoring and detection of NDV in China,can be widely used.