Development Of Recombinase Polymerase Amplification For Aapid A Rapid Detection Method For Canine Parvovirus

Canine Parvovirus disease(CPVD),caused by Canine Parvovirus(CPV),is an acute,highly contagious and lethal infectious diseases.It emerged as a novel pathogen in 1978 in the United States and spread rapidly worldwide,that with clinical manifestations of hemorrhagic enteritis and non-purulent myocarditis.Puppies of 2-8 months harmed serious,which the characteristic of acute onset,short course,strong infectivity and highly mortality rate.Chian was first reportrd CPVD in 1982 and spread rapidly all the country.In China,it is one of the major canine infectious diseases,also affects seriously to the development of the dog breeding industry.Recombinase Polymerase Amplification(RPA)is a new isothermal gene amplification technique.It formed by recombinase and primer complexes on the template to find with the original sequence,trigger a chain of exchange reaction after positioning,start the DNA synthesis,in order to template on the purpose of the fragments of the exponential amplification.It can work well at 25-42degree centigrade,10-15 minutes,which RPA can amplify nucleic acids rapidly.The RPA products can be combined with lateral flow dipstick,biochip,gelelectrohhoresis,etc.RPA is sensitivity,specificity,time-shorter and simple operation.At present,it is the most convenient and fast nucleic acid detection method available for instant disgnosis.To establish a rapid and simple detection method for canine parvovirus(CPV),the recombinase polymerase amplification(RPA)assay was developed with the primers designed according to the conserved sequence of CPV VP2 gene and LFD-RPA also established on this basis.Select the best primers and determine the best reaction conditions.The detection procedure of RPA assay was completed within 15 min at 38℃.Based on pEASY-CPV-VP2 as template,the analytical sensitivity of the RPA and LFD-RPA was 10~1 copies/reaction of a standard DNA template,which was 10 times more sensitive than the common PCR method.The assay was specific for the taeget sequence amplification from CPV,and had no any amplification from other viral pathogens.The positive detection rate of clinical samples with suspected CPV infection using the established RPA and LFD-RPA method was88%,which was significantly higher than the detection rate of conventional PCR(80%)and the detection rate of colloidal gold(76%).The RPA assay established in this study was a simple,rapid,reliable and affordable method that can potentially be applied for the detection of CPV in the research laboratory and on site diagnosis.