RT-ERA Combined With CRISPR-Cas12a System For Rapid Detection Of Establishment Of A Method For The Detection Of Avian Leukemia Virus In Subgroup J

Avian leukosis(Avian leukosis,AL)is a contagious neoplastic disease caused by avian leukosis virus(Avian leukosis virus,ALV).Due to the continuous mutation of Avian leukosis virus subgroup J(VAL-J),which has stronger pathogenicity and faster transmission ability,chickens infected with Avian leukosis virus show reduced production performance and low immunity,which indirectly causes immunosuppression of the body and leads to immune failure of other vaccines,causing a This has caused certain economic losses to the development of China’s poultry industry.At present,the detection methods of ALV-J are all laboratory tests,which require professional personnel and large instruments and equipment,and cannot be used to achieve rapid on-site detection.Therefore,the establishment of a simple,convenient and rapid diagnostic method for ALV-J is of clinical significance to achieve the rapid detection of this pathogen.Enzymatic Recombinase Amplification(RT-ERA)is an isothermal amplification technique that allows nucleic acid amplification in a water bath,saving more time for experiments.with nuclease exocytosis activity,and has become a hot research topic for in vitro diagnostics.Currently ERA combined with CRISPR-Cas12 a system can be used for the detection of various viruses,but whether the combination of the above two methods can be used to achieve the detection of subgroup J avian leukemia virus has not been reported.Therefore,in this study,we proposed to combine RT-ERA amplification with CRISPR-Cas12 a system to establish a fluorescence method and test strip method for detecting subgroup J avian leukemia virus with the gp85 gene of subgroup J avian leukemia virus to provide a new method for early diagnosis and rapid field detection of avian leukemia virus and provide some help for the purification of ALV-J.The specific methods and results of the experiment are as follows:1.Genetic evolutionary analysis of the gp85 gene of three subgroup J avian leukemia virusesThe full-length amplification primers of ALV-J were designed,the gp85 genome was amplified and sequenced,and the recombinant plasmid p MD-19T-gp85 was successfully constructed;the gp85 fragments of the exogenous subpopulation reference strains published in Genbank were analyzed for homology,and the results showed that the nucleotide homology of the gp85 gene sequences of the three ALV-J strains was 84.7-86%.The genetic evolutionary tree analysis showed that all of them belonged to the J group.2.Establishment of RT-ERA method for detection of subgroup J avian leukemia virusThe conserved gene gp85 of ALV-J was selected as the target,and three pairs of cr RNA primers were designed on the gp85 gene to initially establish the CRISPR-Cas12 a reaction system.The PCR amplification product was used as the template to screen the cr RNA primers,and the third pair of cr RNA primers cr RNA-F3/cr RNA-R3 was selected for subsequent experiments.Four pairs of RT-ERA primers designed based on primers cr RNA-F3/cr RNA-R3 were screened for the optimal primers;the RT-ERA was optimized for time temperature;and the sensitivity assay was performed.The results showed that the optimal primers were ERA-F3/ERA-R3;the optimal reaction temperature was 40℃ and the incubation time was 20min;the sensitivity detection result was 103copies/μL,which was more sensitive than the normal PCR.3.Establishment of RT-ERA combined with CRISPR-Cas12 a for detection of subgroup J avian leukemia virusIn this study,RT-ERA combined with CRISPR-Cas12 a was established as a fluorometric method.The protein concentration and cr RNA primer concentration of the fluorometric method were screened and the reaction time was optimized using the RT-ERA amplification product as the template;sensitivity and specificity tests were performed.The results showed that the optimal protein concentration was 250 nmol;the best cr RNA concentration was 500 nmol;the optimal reaction time was 30 min;the sensitivity could detect as low as 1 copies/μL and the specificity was good.The ALV-J assay was performed on 78 clinical samples,and the visualization detected 19 positive results,with a compliance rate of 97.1% with PCR.In order to observe the detection results more visually,this study introduced test strips based on the fluorometric method,and established a test strip method for RT-ERA combined with CRISPR-Cas12 a,using test strips to present the final detection results.In this study,the probe concentration and reaction time of the test strips were screened and optimized;and the sensitivity and specificity of the test strip method were tested,and the results showed that: the optimal probe concentration was 5 nmol,the optimal reaction time was 25 min,and the minimum detection limit of the sensitivity test was 102copies/μL;the specificity test showed no cross-reactivity;78 clinical samples were tested for ALV-J.The results showed that 17 clinical samples were positive,and the compliance rate was 98.7%when compared with the PCR test results.In summary,this study established a rapid diagnostic method for ALV-J using the CRISPR-Cas12 a system.This study demonstrated that the method has the advantages of higher sensitivity,simpler operation and shorter time consumption compared with the common PCR.This study will provide a new method for chicken farms to achieve early diagnosis and rapid detection of ALV-J in the field,and provide some help for ALV-J decontamination.