Study On The Nucleic Acid Detection Method Of Two Typical Pathogenic Microorganisms In Water Environment

With the rapid development of aquaculture industry in China,the prevention and control tasks related pathogenic microorganism are also increasingly intensified.According to the difficulties and market demand in the detection of pathogenic microorganisms,Taura syndrome virus(TSV)and were selected as the research objects.The bioinformatics analysis for the complete genome of TSV was conducted,and the primers were designed based on its conservative sequences.A loop-mediated isothermal amplification(LAMP)method for rapid detection of TSV combined with lateral flow dipstick(LFD)(LAMP-LFD)was established.Furthermore,the merits and demerits of LAMP-LFD technology were compared to real-time fluorescent quantitative PCR(QPCR).For Aeromonas hydrophila,a fluorescence quantitative PCR detection method combined with immunomagnetic bead enrichment for bacteria was established.By optimizing the reaction parameters,the detection specificity and sensitivity were discussed,and the advantages of combining immunomagnetic beads are described.The data will provide significant technical support for the rapid detection of pathogenic microorganisms in aquaculture.The research contents are as follows:(ⅰ)Bioinformatics analysis of Taura syndrome virus.The aim was to make bioinformatics analysis on TSV genein shrimp.The complete genes of TSV were analyzed by bioinformatics software,including its gene sequence analysis,open reading frame prediction(ORF)prediction,physicochemical properties of protein,secondary structure prediction,protein transmembrane and signal peptides prediction,and as well as protein tertiary structure prediction.The TSV gene(JX094350.1)with a length of 10 128 bp was successfully obtained from NCBI gene bank.The bioinformatics analysis showed that TSV gene was a total of 3 286 amino acids,a theoretical isoelectric point(pI)of 5.14,a theoretical molecular mass of 366 443.00u,and an instability coefficient(Ⅱ)37.76,being a stable protein.The complete gene sequence contained two open reading frames(ORFs).There was a transmembrane structure in the protein,and there was not included protein signal peptide.The bioinformatics analysis of TSV is helpful for understanding Taura syndrome virus on molecular level and the prediction of infection mechanism.It will provide useful informations for the prevention and treatment of Taura syndrome.(ⅱ)Establishment of detection method for Taura syndrome virus by loop-mediated isothermal amplification combined with lateral flow dipstick(LAMP-LFD).Taura syndrome virus(TSV)is one of major infectious virus for shrimp,threatening prawn culture.Here,a novel loop mediated isothermal amplification combined with LAMP-LFD method was developed in order to rapidly detect TSV.Three pairs specific primers were designed based on the nucleic acid sequence of viral coat protein 2(VP2)of TSV for LAMP assay.Thereinto,the primer of LF was labeled with biotin(BIO),and LB was labeled with fluorescein amidite(FAM)for lateral flow dipstick(LFD).The results showed that LAMP reaction for TSV was carried out at 63℃ for 40min.The whole detection time was about 45min from LAMP(40min)to LFD(5min).The specificity of LAMP-LFD could be discriminated TSV from other viruses and bacteria.The detection limit of LAMP-LFD was 22.9 fg.Compared to fluorescence quantitative PCR(qPCR),the reaction time was nearly 1 h shorter than that of qPCR.Moreover,LAMP-LFD had high specificity and simple operation without special instruments.It could detect TSV quickly for meeting the needs of fieldbased detection.(ⅲ)Detection of Aeromonas hydrophila in water by immunomagnetic bead enrichment and fluorescent quantitative PCR.As the main pathogenic bacteria of many aquatic animals,Aeromonas hydrophila can cause sepsis.In this study,a rapid detection method of fluorescent quantitative PCR for Aeromonas hydrophila was established by combining immunomagnetic beads for the bacteria enrichment.The primers and TaqMan probes were designed based on its 16S rDNA sequence.The fluorescence quantitative PCR reaction parameters were optimized.And the fluorescence quantitative PCR system was verificated with clinical sample and compared to conventional PCR method.The results showed that the sensitivity of Aeromonas hydrophila was 10 CFU/g using the fluorescent quantitative PCR detection system.Furthermore,by integrating the immunomagnetic bead separation technology,the sensitivity and specificity of the fluorescent quantitative PCR were overall enhanced.In summary,this study was performed bioinformatics analysis on the complete genome of Taura syndrome virus,and successfully established the LAMP-LFD system for TSV detection.Furthmore,A fluorescent quantitative PCR detection method for Aeromonas hydrophila was established by integrating immunomagnetic bead technology.The study on the in-depth understanding of two typical pathogenic microorganism bioinformation and the detection method setting up was of great significance for their related disease prevention and control in aquaculture environment.