Establishment And Application Of RT-RPA Nucleic Acid Dipstick Assay For Detection Of CSFV And PEDV

Classical swine fever virus?CSFV?is the causative agent of classical swine fever?CSF?,which is a highly contagious viral disease that causes large economic losses to farmers all around the world.Porcine epidemic diarrhea virus?PEDV?is the causative agent of an acute,highly contagious and severe enteric disease that leads vomiting,diarrhea,dehydration and high mortality rates in suckling piglets,which seriously inhibit the development of animal husbandry.Therefore,rapid,simple and accurate diagnosis of CSFV,PEDV are critical for agricultural economic and the implementation of effective diseases control measures.Traditional detection methods of CSF and PED are time-consuming,cumbersome,cross contamination and low positive detection rate.Molecular biological detection methods,such as reverse transcriptase polymerase chain reaction?RT-PCR?,have higher requirements on equipment and technical personnel,thus limiting its application in veterinary clinical detection.Recombinase ploymerase amplication?RPA?is a novel isothermal nucleic acid amplification technique,which could complete target nucleic acid amplification in a short time by simulating DNA replication in organisms without temperature cycle.It has the advantages of high specificity,strong sensitivity,simple reaction procedure and needless expensive instruments.Nucleic acid dipstick assay based on flow tomography technology and immune colloidal gold technology.This technology makes it convenient,efficient,rapid and easy to detect the pathogeny.In this study,we developed a reverse transcription recombinase ploymerase amplication?RT-RPA?assay coupled with a nucleic acid dipstick assay for CSFV and PEDV.The method can complete the amplification under 37?within20 minutes,compared with the traditional detection method the established methods have the advantages of rapid,sensitivity and specificity.At the same time,the evaluation of two detection methods in specificity,sensitivity and feasibility in clinic were tested.The results of preliminary application of the clinical material showed that the established methods have certain clinical application value.We detected CSFV,PEDV,porcine parvovirus?PPV?,pseudorabies virus?PRV?,porcine reproductive respiratorysyndrome virus?PRRSV?,porcine circovirus type 2?PCV-2?,Japanese encephalitis virus?JEV?and E.coli.The results showed that no false positive results were observed indicating the methods were highly specificity.Sensitivity of this method for detection of cDNA obtained from genomic RNA of CSFV was 1 pg,10-fold more sensitive than RT-RPA combined with gel electrophoresis and the conventional RT-PCR method.The minimum of this assay for CSFV detection was 10^2.5TCID₅₀/mL and 10³ copies/reaction of recombinant plasmid,similar with RT-RPA combined with gel electrophoresis and the conventional RT-PCR method.The limit of this assay for PEDV detection was 1 pg cDNA,10³ copies/reaction of recombinant plasmid,10times higher than RT-RPA combined with gel electrophoresis and the conventional RT-PCR method.The sensitivity of PEDV detection was 10².5.5 TCID₅₀/mL,similar with RT-RPA combined with gel electrophoresis,and 10 times higher than conventional RT-PCR method.Furthermore,the diagnose accordance rate of the two established methods compared with RT-PCR detection methods in clinical samples for CSFV?37?and PEDV?38?was 73.5%,84.5%.To sum up,the establishment of CSFV,PEDV RT-RPA nucleic acid test strip method has the characteristics of high specificity and strong sensitivity,and has a certain clinical feasibility.At the same time,in order to avoid the false positive conditions caused by cross contamination of the amplification product and external leakage,the cross contaminated proof cassette was used to visualize the amplification products.The sealing device avoid aerosol contamination,realized the visual detection of the pathogen,and more suitable for application under field conditions.