Study On The Development Of Rapid Detection Methodsof Subgroup K Avian Leukosis Virus

Avian leukosis(AL)is a class of infectious diseasecaused by avian leukosisvirus(ALV).ALV known to be capable of cas uing some hematopoietic cell hyperplasia and induing a variety of tumors.Nearly three decades,avian leukosisvirushave been spreading rapidly around the world,which had causedgreat economic losses for poultry industry.Avian leukosisvirus can be divided into endogenous and exogenous virus.ALV-A,B and J are the most commonexogenous subgroups that mainly cause lymphocytic leukosis and myeloid leukosis.ALV-E isan endogenous leukosis virus,whichhas low or no pathogenicity to chickens directly.Up to now,we have developed many methods to against the disease and have been widely applied to clinical detection,purification of flocks and the commercial test kit is available.However,ALV-K,as a new strain isolated in 2012,we have no effective method for detecting ALV-K.Therefore,the establishment of areal-time RT-PCR and RT-LAMP for detecting ALV-K is of great significant andit will fill gaps in this field.Real-time RT-PCR is a method for detecting ALV-K.Refer to the gp85 geneseq uences of ALV-K which from the GenBank database(GenBank acc.nos.KP686142.1),we designed a pair of specific primers and a specificprobe.It can amplify ALV-K specifically,and unable to amplify other subgroup of avian leukosis virus.Set up positive recombinant plasmid,construct a standard curve.Under optimal reaction conditions,the res ults showed that there was a good correlation between copy number and Ct value of recombinant plasmid.(y=-3.402x+48.355,R~2=0.998,efficiency=0.96768).The detection limits was3.4×10~1copies/uL of positive recombinant plasmid,which was 100 times more sensitive than conventional PCR.The coefficient of variation for intra-and inter-assay was both less than 5%.Inoculate 1-day-old SPF chickens with 4×10~4TCD50 ALV-K via absolute quantification method,compare with the standard c urve of positive recombinant plasmid,we can acquired the template copy number of the organizations to obtain organs.The results indicated that ALV-K were found in heart,liver,spleen,lung and kidney.At the same time,the virusloadin the liver,kidney and spleen was muchhigher than those in the heart and lung.It laid a foundation for further study of the interaction between virus and the tissue,and its detection.RT-LAMP for detectingALV-K is a method that designed a pair of specific inner primers(FIP/BIP)and outer primers(F3/B3)in highly conserved regions.It can be specifically amplified the ALV-K just at the concentration of the primers and the o uter primers were 1.6uM and 0.1uM,the reaction temperature is 61?,constant temperature for60 minutes,and the reaction to achieve the minimum Ct value and maximum fluorescence.RT-LAMP reaction is able to detect nucleic3.4×10~4copies/uL at least.According to theRT-LAMP detected method,it has not been fo und ALV-K among the 40 chickens which are randomly select for detecting from the chickens supply to Macau.The results from the detection are consistent with the ordinary PCR.RT-LAMP detected method is simple,short reaction time,low requirement for detected instrument,low cost,and has great clinical significance.Conclusions as a result,real-time RT-PCR and RT-LAMP of ALV-K is a fast,effective specific and accurate testing method which co uld be widely used in laboratory and clinical testing.Thisstudy provide a powerful technical support for theeradication program of avian leukosis.