Development Of Rapid Detection For Peste Des Petits Ruminants Virus By Loop-mediated Isothermal Amplification Method

Peste des petits ruminants(PPR)is an acute and highly contagious viral disease of sheep.Goats can be particularly seriously infected,Wild animals can also be infected. Clinical characterization of which is pyrexia,ocular and nasal discharges,erosive stomatitis and diarrhoea.The morbidity and mortality rates of PPR can be as high as 100%and over 90%.Respectively,According to the notice of the Ministry of Agriculture,in July 25th of 2007,the first outbreak of PPRV disease in Long Menka village of Tibet was confirmed,which caused 371 infection,262 goats diad and 522 goats were killed,which caused serious economic losses.So development a fast sensitive and efficient method for PPRV detection has important significance. Loop-mediated isothermal amplification method(LAMP)has been wildly used in pathogen detection for its easy-implementation,fast,efficient and sensitive characteristics.Up to now,PPRV-LAMP method hasn't been reported in china.so this research has not only application values but also scientific values.The nucleocapsid protein of PPRV is the major structure proteins,which induces important biofunctions and plays important roles in the pathogenic process and immune response.It is a good target for PPRV differential diagnosis.In this research, the partly conserved sequence within regionâ…£of N gene of China/Tibet/0701 PPRV was selected by bioinformatics method to design LAMP primers.Optimal primers were designed using bioinformation software(Primer Explorer V3),according to some important parameters including:GC content,5'-3'-term stability,secondary structures and so on.Because the amplification product of LAMP are a series of diffuse bands,which was difficult to distinguish when non-specific amplification product appeared.In order to verify the specificity of the response production,restriction sites EcoRâ… and EcoRâ…¤were also designed in inner primers to ensure the reliability of the experiment.The result of amplification us conserved primers showed that the third loop primers have the best amplification efficiency.Furthermore,reliability has been proved by enzyme digestion.The LAMP method for PPRV detection was set up by using pMD 19-T-N plasmid that contains N gene of China/Tibet/0701 PPRV.In the following steps,several important parameters of LAMP were optimized including temperature,concentration of Mg²⁺,Betaine,dNTP,Bst DNA Polymerase and LAMP primers,etc.The results indicated that Mg²⁺,dNTP and Bst DNA Polymerase have synergy effect.The best result was achieved with a dNTP concentration of 0.4 mM,Mg²⁺ concentration of 6 mM,Betaine concentration of 1.6 M,Bst DNA Polymerase concentration of 8U.Based on the optimized primers,the PPRV-LAMP method was developed.A set of time-consuming,sensitivity,specificity and reliability tests have been done, compared to a standard PPRV-PCR detecting method recommended by OIE.It has been proved by time-consuming test that only 40 min would be taken by PPRV-LAMP to get positive result.In sensitivity test,the detection limit was 1.6 TCID₅₀ of PPRV template which was validated by using serial diluted templates.The sensitivity of PPRV-LAMP was 10 fold of the OIE standard PCR method;Specific experiment showed that there were no non-specific amplification occurs using Measles virus,Canine distemper virus,Newcastle disease virus,Avian influenza virus and Parainfluenza virus genomes as templates.In repetitive and stability tests,experiments with 10 batches of Nigeria 75/1 and China/Tibet/0701 PPRV templates and 10 experiments with the same templates have been done.Stable outcomes guaranted the reliability of PPRV-LAMP method.To sum up,a rapid,sensitive,efficient and reliable PPRV-LAMP method has been developed in this research,which is a firm foundation of the following development of PPRV-LAMP rapid assay kit with high application value.