Research On The Rapid Detection Technology Of ASEA Nucleic Acid For Pet Viruses Such As Canine Parvovirus

Canine,as a kind of popular animal,can not only accompany human in daily life,but also play an important role in social security and anti-drug work,so the protection of the dogs' health and the prevention of fatal canine virus infections are extremely important.Canine parvovirus(CPV)and Canine coronavirus(CCo V)are common pathogenic viruses with strong infectivity in canine,and rapid detection of these viruses are of great significance.Although colloidal gold test strip and other convenient detection methods have been developed,most of these methods which are based on antibody detection could hardly detect pathogens during the incubation period.Considering the canine training centers or kennels need more sensitive detection methods to prevent the rapid spread of the virus,it is essential to establish a simple and rapid detection method for CPV and CCo V.The accelerated strand exchange amplification(ASEA)as a rapid detection method was used to detect CPV in this article.The experiments of feasibility,temperature optimization,specificity,anti-interference,and sensitivity for CPV detection are researched.The results show that the optimal ASEA primers for CPV detection have the highest amplification efficiency at 61 ? and 74 ?.The primers have good specificity and anti-interference,which can distinguish CPV DNA from genomic nucleic acids of canine,E.coli and 8 kinds of common canine viruses,and can detect CPV DNA from the nucleic acid mixture.The limit of detection for CPV detection with ASEA is 4.8 copies/?l,and the peak time is 16.6 min.Combining with the improved 3 min pyrolysate method,the whole detection process is completed within about 20 min.Based on the established ASEA for detecting CPV genomic DNA,the article tests the actual samples of CPV.Using ASEA,real-time PCR and colloidal gold test strip to detect 42 sick fecal samples of canines with diarrhea and vomiting symptoms,comparing the accuracy of the three methods on the actual samples.The results shown that ASEA and real-time PCR detect 19 cases of CPV infection,while colloidal gold test strip detect16 cases of CPV infection.The agarose gel electrophoresis of ASEA and real-time PCR amplification products finally prove that these three samples were infected by CPV.Therefor,the detect result of ASEA and real-time PCR is accurate,and the colloidal gold test strip detect three false negative samples.We speculate that the reason for this phenomenon is that these three samples are in the early stage of CPV infection.The antibodies in the infected dogs are not enriched to the concentration detectable by colloid gold test paper,while the viral nucleic acid could be detected by ASEA and real-time PCR in the early stage of virus invasion.In conclusion,the detection rate of ASEA for CPV is consistent with that of real-time PCR,which is better than that of colloidal gold test strip.Compared with real-time PCR,ASEA has obvious advantages in time.And ASEA also has advantages in early diagnosis compared with colloidal gold test strip.Based on the above research of CPV detection by ASEA method,this research project cooperates with Qingdao Jianma Gene Technology Co.,Ltd.to develop ASEA nucleic acid detection kits for CPV and CCo V.In the follow-up of this project,two kits are used to detect 42 sick fecal samples.The detection results of CPV are consistent with prior research content,and the detection results of CCo V are consistent with colloidal gold test strip.And after testing by the detection kit and colloidal gold test strip,two samples are detected with CPV and CCo V at the same time,which confirming that CPV and CCo V can co-infect the host.In summary,two ASEA nucleic acid detection kits for detecting CPV and CCo V have good performance.The kits are suitable for the market and have great potential.