Detection And Pathothying Of Newcastle Disease Virus By SYBR Green I Real-Time RT-PCR

Newcastle disease (ND) is a highly fatal and highly contagious avian disease caused by Newcastle disease virus (NDV). It is also one of the List A animal infectious diseases, and causes severe avian epidemics worldwide every year. There are several kinds of classical NDV detection methods, such as virus isolation, HA and HI test, immunofluencent test, ELISA, etc. These techniques some are time-consuming, some are insensitive or somehow unspecific.Common RT-PCR can detect a few copies of nucleic acids of NDV, and therefore it is used to detect NDV for clinical or quarantine purpose. But its uses are quitely limited because it is difficult to avoid the false positive signals caused by the pollution of PCR products. Common RT-PCR also requires the uses of EB, a strong carcinogen.Real-time RT-PCR assay is one of the new detection techniques. It has high sensitivity as common RT-PCR, and avoids both the pollution of PCR products and the uses of EB. It is also faster than common RT-PCR. SYBR Green I releases fluorescence when combining to double-stranded DNA, and based on this principle, SYBR Green I real-time RT-PCR is applied widely with lower cost and without difficult probe design. It is suitable for the detection of highly variable genes.Here in this paper SYBR Green I real-time RT-PCR techniques are reported to be established to detect and pathotype NDV.Firstly, many sequences of NDV F gene were analysed and a general pair of primers were designed to detect all NDV strains using SYBR Green I real-time RT-PCR technique. Then, the detection conditions were opimised, and RT and PCR were integrated into one step. The Tm values of the specific amplification products are always (88+1)C. The results of such assay and the sequencing assay for the detection of 10 unknown strains were the same, and the sensitivity of such assay were higher than a reported common RT-PCR assay.The virulence of NDV strains are determined by the lysis site sequences of the viral F protein. Based on this principle, two pairs of primers were desgined to correspond to the sequences of the F protein lysis site of virulent NDV strains and avirulent strains, respectively. The viral RNA was detected using the two primers respectively, and the virulence of NDV strains were judged according to the amplification effects of the twopairs, of primers. The results of such assay and MDT assay for the pathotyping of 8 unknown strains were the same, which suggested that this assay can be used to pathotype NDV strains and better than the common RT-PCR technique reported previously. The amplification products of the pair of aforementioned general primers could be used as the template of such pathotyping RT-PCR assay, and the high sensitivity of the aforementioned assay of general NDV detection was inherited thereby.The real time RT-PCR techniques report here are of important significance in veterinary clinicals and the quarantine of avian product export and import.