Complete Genome Sequence Analysis And Establishment Of RPA-LFD Detection Method Of Black Queen Cell Virus

Objective In this study,a strain of Black Queen Cell Virus(BQCV)complete genome was isolated from Jinzhou,Liaoning Province and named as BQCV-LN2022(Gen Bank: OQ799601).The whole genome was cloned and its gene sequence was analyzed.It lays a foundation for the study of BQCV genetic background.On the previous study,highly conserved segments of the structural protein gene of BQCV-LN2022 were selected as target genes,with Recombinase Polymerase Amplification and Lateral Flow Dipstick technique.The RPA-LFD detection method of BQCV was established.To provide reference and technical reserve for prevention and treatment of BQCV.Methods After screening and purification,RNA was extracted from the collected diseased material,and detected using RT-PCR technology.The result was confirmed to be positive for BQCV,which was named BQCV-LN2022.Referring to the gene sequence of BQCV-JL2016(MZ821803.1),13 pairs of primers were designed and synthesized using Primer Premier 6.0 software to clone the entire gene of the isolated BQCV.Using non structural protein genes and structural protein genes as target genes,the sequence of 26 BQCV isolates from different regions included in Gen Bank were compared in homology.A phylogenetic tree of BQCV was constructed using MEGA7.0 to study the genetic and evolutionary relationships of the isolates.By studying the highly conserved structural protein region(7903～8322 bp)of BQCV-LN2022,RTq RPA specific primers and probes were designed,and a RT-q RPA detection method for BQCV was designed.By optimizing the combination of primer and probe pairs and establishing a standard curve,the specificity,sensitivity,and repeatability of the method were evaluated.Results The genome of BQCV-LN2022 isolated in this study has a total length of8449 bp and contains two Open reading frame(ORFs).The ORF1 encoding non structural proteins is located at 642-5528 bp of the entire gene,while the ORF2 encoding structural proteins is located at 5845-8298 bp of the entire gene.The results of homology analysis showed that the non structural protein nucleotide Sequence homology of BQCV-LN2022 was 87.4%～97.9%compared with the other 26 BQCVs,the highest homology with SH2019 strain,97.9%,and the lowest homology with Australia Sydney strain,87.4%.The structural protein Sequence homology of BQCV-LN2022 was 89.4%～98.8%,the highest homology with JL2016 strain,reaching 98.8%.The homology with the Australian Sydney strain is the lowest,at 89.4%.The box chart shows that the overall distribution of Sequence homology values of BQCV-LN2022 structural protein is more concentrated than that of non structural protein.Although non structural proteins have lower homology and greater differences compared to structural proteins in nucleotide sequences,the overall distribution of homology values between structural and non structural proteins in amino acid sequences is not significantly different.A developmental evolution tree was constructed using the non structural protein of BQCV as the target gene,forming two branches.FJ2019 and SH2019 belong to the same sub cluster,while BQCV-LN2022 and them belong to the same branch.Using structural proteins as the target genes for genetic evolution analysis,the evolutionary tree has also formed two branches SD2019 and HN2019 belonging to the same cluster,while BQCV-LN2022 belongs to the same branch as them.Predicting the N-type glycosylation sites of BQCV-LN2022,three possible glycosylation sites were predicted,located at 112 aa,314 aa,and 577 aa,respectively.The optimal RPA primer pair and probe combination were obtained through screening.The amplified target fragment was 199 bp,with an optimal reaction temperature of 37 ℃ and a reaction time of 30 minutes;The lower detection limit is 1.26 × 101 copies/ μL;The specificity results showed that only BQCV showed a specificity curve and did not cross react with other common bee viruses.Showing good specificity and sensitivity.Afterwards,RPA technology and LFD(side flow chromatography test strip)technology were combined to make BQCV pathogen detection easier to distinguish,and a rapid RT-RPA-LFD detection method was established.Its detection temperature range is 20 ℃～50 ℃,with a minimum detection time of 5 minutes and an optimal detection time of 15 minutes.The detection limit is 1.26 × 101copies/ μ L.Using this method to test 24 clinical samples,21 positive samples were detected,which is the same as the results of RT q PCR detection.Therefore,it can be used as an effective tool for clinical testing.Conclusions1.The complete gene sequence of BQCV-LN2022(Gen Bank: OQ799601) was successfully obtained.The nucleotide sequence of its structural protein had the highest homology(98.8%)with JL2016 strain(MZ821803.1),and the lowest homology(89.4%)with Australia Sydney(MW390818.1).The nucleotide sequence of non structural protein has the highest homology with SH2019(MZ821815.1),reaching 97.9%.It has the lowest homology with Australia Sydney(MW390818.1)at 87.4%.And BQCV-LN2022 belongs to the same evolutionary cluster as FJ2019(MZ821801.1)and SH2019 strain(MZ821815.1).Predicting the N-type glycosylation sites of BQCV-LN2022,three possible glycosylation sites were predicted,located at 112 aa,314aa,and577 aa,respectively.2.Successfully established RPA-LFD detection method for the Black Bee Queen’s Nest virus,with a detection limit of 1.26 × 101copies/ μL.The minimum detection time is 5 minutes,the optimal detection time is 15 minutes,and the detection temperature range is 20 ℃ ～50 ℃.And it has no cross reaction with other common bee viruses,has good sensitivity and specificity,and is shorter in duration,easy to operate,and results are easy to visualize and visually judge,suitable for on-site detection and promotion of BQCV application.