Establishment And Application Of Quantitative Fluorescent RT-PCR For Detection Of Newcastle Disease Virus

Newcastle disease（ND）is caused by the strong strain of Newcastle disease virus（NDV）.At present,the NDV epidemic in our country has many genotypes,a wide host range and a wide contamination area.The existing fluorescent RT-PCR detection technology in"NDV Diagnostic Technology"（GB/T 16550-2020）can only be used to evaluate strong NDV strains,and cannot meet the demand for rapid detection of all the NDV genotypes prevalent in China.At present,the detection rate of NDV epidemic strains detected by the domestic fluorescent universal RT-PCR detection kit is only 85%,with some missing detection phenomenon.Therefore,it is necessary to establish a rapid,sensitive and efficient specific molecular detection method that can cover all the genotypes of NDVS currently prevalent in China.Two sets of specific primer probes were designed for NP genes and P genes of all the domestic popular NDV genome sequences,and the optimal primer probe combination was selected.A universal fluorescent quantitative RT-PCR detection method for NDV was established,and a standard curve was drawn to evaluate the repeatability,specificity and sensitivity of the method,and to determine the positive judgment value.The results show that the standard curve equation of this method is Y=-3.512X+18.716,and the correlation coefficient R₂is 0.996.The method had strong specificity and no cross reaction with AIV（H5subtype,H7 subtype,H9 subtype）,IBV,FAV,IBDV,Pi HV and ILTV.The detection limit was103～105 EID₅₀/0.1m L,which was 10 times higher than the conventional RT-PCR method.The coefficient of variation of intra-and intergroup repeatability tests were 0.98～0.26 and1.07～0.19,respectively,which were both less than 3%.Three clinical samples were diluted with multiple ratio of nucleic acid and tested.Finally,the positive determination value of fluorescence RT-PCR method was determined to be Ct value≤37 and the typical amplification curve appeared.1044 clinical samples（916 oropharyngeal/cloacal swab samples and 128 NDV-infected tissue samples）were tested using the universal fluorescent RT-PCR detection method established in this study,and compared with the results of virus isolation and identification（gold standard）.The Yoden index was calculated and ROC curve was drawn to complete the evaluation of various diagnostic indicators.The results showed that 58 NDVS were isolated from 916 oropharyngeal/cloaca swab samples,including 25 NDVS in class I and 33 NDVS in Class II.128 NDV-infected tissue samples were prepared,and 91 samples were positive after virus isolation and identification.Fluorescent RT-PCR was performed on 1044 clinical samples and compared with the results of virus isolation and identification.The area under ROC curve drawn was 0.9718,indicating high diagnostic accuracy of the method.When cutoff value is 37,the Yoden index is 91.61%,the diagnostic sensitivity is 92.62%,and the diagnostic specificity is 98.99%.The coincidence rate between the method and the"gold standard"is 98.08%,and the Kappa coefficient is 0.915.In conclusion,the method established in this study has strong specificity,high sensitivity,good repeatability and high diagnostic accuracy,which is suitable for the high throughput rapid detection of Newcastle disease virus.