Rapid Detection Of Newcastle Disease Virus By Reverse Transcription Loop-mediated Isothermal Amplification

Newcastle disease is an important poultry infectious disease, gives serious damageand difficult to prevent and control. Reverse transcription loop-mediated isothermalamplification (RT-LAMP) and the LAMP real-time detection technology for Newcastledisease were developed by us. It is a fast, accurate qualitative or quantitative method.This method is expected to replace the existing Newcastle disease virus nucleic aciddetection, implementation of Newcastle disease quickly confirmed. This technology canprovide technical support for the effective prevention and control of Newcastle disease.The study consists of three parts:Part I: Cloning and sequence analysis of Newcastle disease virus Fusionprotein gene (F gene)According to the F gene sequence of Newcastle disease virus strains in GenBank,one pair of primers were designed and synthesized. The amplified fragment fromRT-PCR was cloned into vector pGEM-T. Recombinant plasmid pGEM-T-NDV wasobtained after transformation and screening. F gene of Newcastle disease virus wasobtained after the identification by restriction endonuclease digestion, PCR andsequencing.Part II: Development of RT-LAMP detection method for NDV.Six primers were designed and synthesized according to the NDV F gene.Reaction temperature, reaction time and [Mg2+] concentration in mixture wereoptimized and the effective RT-LAMP method was developed. After amplification, themixture was analyzed by electrophoresis or observed with naked eye directly. In gelelectrophoresis analysis, multi-strip feature of the amplification product was observed.In naked eye observation, reaction product was turbid. The whole reaction time is about 0.5h and100times more sensitive than RT-PCR. RT-LAMP method for Newcastledisease virus developed by us has many merits, very quickly; do not requiresophisticated equipment, high sensitivity and specificity, simple operation and so on.Part III: Real-time quantitative analysis of Newcastle disease virus by LAMP.Real-time turbidity in RT-LAMP of Newcastle disease virus was detected and thequantitative method for Newcastle disease virus was established. Whole detection wascompleted in0.5h. Powerful linear relationship was found between the copy number oforiginal plasmid DNA template range from1.98x108to1.98x104and the time ofturbidity under0.1. Dose of virus can be evaluated. It was also confirmed that the ringprime can improve reaction rate in LAMP reaction. After the stability and specificvalidation, it was confirmed that the real-time turbidity LAMP detection method is areliable, effective, accurate and operation simple method.