Application Of PCR Technology Included With Iac In The Detection Of Dogs And Cats Five Viruses

PCR technology has been constantly innovated and developed scine Kary Mullis invented PCR technology.PCR can be used as an in vitro amplification technique to amplify specific DNA fragments in a short time.The method has the characteristics of strong specificity,high sensitivity and simple operation.The amplified products can be detected by agarose gel electrophoresis.The DNA bands can been seen under the ultraviolet.PCR technology are widely used in the detection of disease,pathogen,microorganisms.However,if there are PCR inhibitor,PCR instrument failure,PCR reaction system error,polymerase inactivation and so on during PCR detection,it will cause the false negative result.It is difficult to find it by positive control and negative control.Therefore,it is necessary to add a control that can indicate false negative results in the PCR reaction system.In order to solve this problem well,the researchers introduced the amplified internal control.The amplified internal control(IAC)is a non-target DNA sequence added to the PCR assay system,which could be amplified with the target DNA sequence at the same time.The constructed DNA fragment or housekeeping gene fragment which was not homologous with target DNA sequence can be used as IAC.IAC is equivalent to a control signal and can be always amplified although there may be no target sequence.Therefore it can be used to indicate false negative phenomenon.In this reaearch,PCR technique with amplified internal control was studied on the diagnosis of 5 common infectious diseases(canine distemper,canine parvovirus,Feline panleukopenia,Feline infectious peritonitis,and Feline infectious rhinotracheitis)of dogs and cats.Establishment the pcr detection method for five infectious diseases of dogs and cats1.Establishment the PCR detection method for five infectious diseases of dogs and catsAccording to the canine distemper virus F gene,canine parvovirus VP gene,feline panleukopenia virus conserved gene,feline infectious peritonitis virus conserved gene,feline infectious rhinotracheitis virus gD gene sequence,design 5 pairs of specific primers,set up five kinds of PCR detection method for communicable diseases of cats and dogs.The results showed that the canine distemper virus primers CDV-337F/R could specifically amplify the target gene fragment of canine distemper virus 337 bp from canine four-combination vaccine.The canine parvovirus primers CPV-448F/R could specifically amplify the target gene fragment of canine parvovirus 448 bp from four-combination vaccine.The feline panleukopenia virus primers FPV-391F/R could specifically amplify the target gene fragment of feline panleukopenia virus 391 bp from the cat three combination vaccine.The Feline infectious peritonitis virus primers FIP-223F/R could specifically amplify the target gene fragment of feline infectious peritonitis virus 223 bp from the cat infective peritonitis case.The feline infectious rhinotracheitis virus primers FHV-467F/R could specifically amplify the target gene fragment of feline infectious rhinotracheitis virus 391 bp from the cat three-combination vaccine.2.Establishment the PCR detection method with the internal control for five infectious diseases of dogs and catsIn this study,duck microsatellite AMU70 was used as internal control gene,and five kinds of amplified internal standard plasmids were constructed by compound primer recombination method.The five kinds of amplified internal standard plasmids had the same primers as five kinds of virus PCR detection.Five kinds of Internal amplification control plasmids were optimized to establish PCR detection method for 5 kinds of virus containing amplified internal standard plasmids.The best results showed that in the 20 ?L reaction system,canine distemper virus detection by adding the internal standard was 105 copies,Internal amplification control plasmid and target gene amplification effect is better.canine parvovirus virus detection by adding the interlal control was 106 copies,feline panleukopenia virus detection by adding the inteCMal control was 105 copies,feline infectious peritonitis virus detection by adding the inteIla control was 107 copies,feline infectious rhinotracheitis virus detection by adding the interna control was 107 copies.In summary,after the clinical test results show that the establishment of this experiment with PCR technology included with IAC method can quickly and accurately,simply detect the above dogs and cats five diseases,and can effectively avoid false negative phenomenon,to laid a solid foundation of infectious disease early molecular diagnosis.