Development Of Loop-mediated Isothermal Amplification Methods Targeting The FneB Gene For Detection Strangles

Objective:Developed a novel LAMP method（fne B-LAMP）to detect the fne B gene of Streptococcus equi subsp.equi（S.equi）,the causative agent of strangles in equids.Detection specificity and sensitivity and optimize the LAMP detection method to establish a quick and simple LAMP method.This method can provide a theoretical basis and methods of operation for primary LAMP detection for strangles.Method:LAMP primers were designed that targeted fne B gene from Streptococcus equi subsp.equi 4047complete genome,the sequence of repetitive element（accession number:FM204883.1）was downloaded from the NCBI Gen Bank.The sequence was further analysed by software.The DNA template used in this study were 2 strains of S.equi from Boiling method,and amplified the target sequence of the fne B gene at63℃within 60 min to evaluation of primers.The reaction conditions were optimized including temperature,time and Mg²⁺concentration etc.The total DNA of several heterologous parasites were extracted from Staphylococcus aureus,Streptococcus agalactiae.Escherichia coli,Bacillus cereus,Streptococcus to assess the specificity of the LAMP assay;DNA was extracted from two SEE strain DNA templates were prepared by a serial 10 fold dilution with concentrations.For the purposes of comparison,using PCR with the same amount of DNA templates were also performed.The PCR were used to screen 38 nasal swabs and 2 submandibular lymph nodes pus-like obtained during an outbreak of strangles.By culture examination and targeted amplification of fne B gene for Streptococcus hemolytic streptococcus,and analyze sequencing results and compare homology.Sequencing one rod-shaped isolate that also produces beta hemolysis and one alpha hemolytic streptococcal strain.The isolated strains subjected to sequencing analysis were detected by the optimized fne B-LAMP method.Results:Reactivity of the fne B-LAMP:The reaction successfully amplified the target sequence at 63℃within 60 min and LAMP mixture was made in 25μL of reaction mixture containing 5 M betaine,0.2 m M of each d NTP,40 m M Mg²⁺used water bath.It was proved that the positive result only appeared when S.qui were used as templates,but not with the negative control（double-distilled water）or the other 5 strain.The detection limits of the reaction for template DNA was 8 pg/μL and the detection limit was>4 times higher than that for PCR.It was proved that the positive result when 7 isolated strains subjected to sequencing analysis were detected by the optimized fne B-LAMP method,and can be used in test strangle.Cloning and sequencing analysis the targeted amplification of fne B gene of beta-hemolytic streptococci.Isolation and identification 6 strains of SEE from 40 clinical samples,and fragments of thesame length were also found 1 SEZ.The 4 SEE strains isolated from nasal swabs from the same horse farm that nucleotide homology from 96%to 99%,and homology of amino acids is 96%～99%;High homology with SEE strain isolated from pus of Submandibular lymph nodes.The Streptococcus equi subsp.zooepidemicus stain had the highest homology with accession number FM204884.1.The sequencing results of another beta-hemolytic rod-shaped bacillus were bacillus cereus and alpha hemolytic strains.Conclusion:This experiment proved fne B-LAMP method is more sensitive than PCR,rapid,simple,and can be used in test strangle;Targeted amplification of fne B gene can detect SEZ strain from clinical strangle and S.equi can be more easily assigned from Submandibular lymph node pus.