| As a highly acute .contagious, septic infectious disease, Newcastle virush has brought severe injury to poultry breeding.Traditional diadynamic methods mostly based on the separation and iditification of the virus and the serological detetions includes Neutralization ,Agar diffusing and Enzymelink-ed immunoadsordent assay,etc.But there are some deficiencies of time-consuming,low sensitivity and depending gracvely on experience.PCR is a quick and accurate assay of detecting NDV,but its amplifictor need to be electrophored in the agrose and stained by Ethidium bromide which can cause human cancer.and the pollution and false-positive result are always caused .So the quick,accurate and highly efficient assay to detect NDV is needed for the Modern production.Real-time fluorescent PCR is a new technology of detecting nucleic acid,It is already greatestly applied to detect bacterium,virus and inherited disease. Basing on the principl of PCR ,this study created fluorescent real-time PCR detection of Newcastle Disease virus of chickens using fluorescent double-stranded primers.There three parts in this study:Patr â… Cloning of the gene of F-Glycoprotein of the Neweastle Disease virus a pair of specific primers(P1:5'-CTT GAC ACC TCA TAC TGT AT-3',P2:CCC AAG CCA TAA TAA GGT CTT-3')were designed based on the mRNA of F-Glycoprotein of Neweastle Disease virus.RNA of the NDv was extracted and reverse transcripted.The production of reverse transcription was amplified by PCR using the designed primers.The electrophore of the PCR production in 1% agarose indicated that the amplifictor is about 623bp.The amplifictor is purified and joined into the pMD 18-T Simple Vector,then transformated into the E.coli JM109.After screened by selecting white spot and blue spot ,then evaluated by PCR ,cloned plasmid was sequenced .The results showed that the sequence of F-Glycoprotein of Neweastle Disease virus has homology of 99 % compared to other strains.Part â…¡ The consructon of real-time PCR detection of Newcastle Disease virus of chickens using fluoresc- ent double- stranded primers a pair ofpositive primers were designed based on the mRNA of the F-Glycoprotein of the Neweastle Disease virus.At the same time ,the negative primers desig- ened basede on positive primers can comple- tely combine with positive pri- mers.FAM was labelled on the 5'end of the positive primers and DABCYL was labelled on the 3' end of the negative primers .When positive primers were hybridized with negative primers ,the flour- scence generated by FAM was quenched by ABCYL.Then the fluorescent double stranded primers were formed, and they can be used to detecte NDV and other 4 species virus including of ATV,ILTV,IB V and G .The experiment showed that no signal was detected for the others except NDV. The experiments showed that the sensiti- vity of real-time PCR is 1000 times highercontrast to common PCR.Part â…¢ The application of Fluorescent real-timePCR to detect NDv quickly DNA templates which concentrationwas know were amplified utilizing the fluorescent real-time PCR.and Ct was analysised by software . 12 samples was detected quantitatively using the fluorescent real- time PCR. 126 samples which from poultry farms of qingdao and weif- ang and jining and zaozhuang were detected utilizing the methods of fluorescent real-time PCR , common PCR and HA(HI) respectively. The result indicates that the positive rate of ND is 61.11%(77/126) and 51.59% (65/126) and 10.32%(13/126) respectively.
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