Establishment Of LAMP Detection Method For Staphylococcus Aureus,escherichia Coli And Staphylococcus Epidermidis

Background:Bovine mastitis is one of the diseases that seriously endangers the dairy farming industry,and at the same time poses a serious threat to the safety of dairy products and human public health.The main pathogenic bacteria that cause mastitis inculde Staphylococcus aureus,Escherichia coli and Staphylococcus epidermidis.Rapid detection of the main pathogenic bacteria of dairy cow mastitis is an important guarantee for early diagnosis and treatment.However,traditional pathogen detection methods have problems such as labour-intensive,time-consuming,requiring sophisticated instruments and professional inspectors,which are not suitable for on-site rapid detection of mastitis-causing bacteria in dairy cows.Therefore,it is crucial to develop a rapid and accurate field detection method for pathogen detection.This study intends to establish a loop-mediated isothermal amplification technology(LAMP)for three pathogens of Staphylococcus aureus,Escherichia coli and Staphylococcus epidermidis,in order to provide technical support for the rapid detection of the pathogenic bacteria of dairy cow mastitis.Purpose and methods:In this study,taking Staphylococcus aureus nuc gene,Escherichia coli fecA gene and Staphylococcus epidermidis SesB gene as target genes.Firstly,multiple sets of LAMP primers designed were screened,and the major factors of the reaction such as Mg2+,betaine,dNTPs,time and temperature were further optimized.Then,the specificity and sensitivity of the established LAMP detection method were evaluated.Furthermore,the established LAMP detection method was used to detect clinical samples of dairy cow milk samples.The results are shown as below:1.The nuc gene of Staphylococcus aureus,fecA gene of Escherichia coli and SesB gene of Staphylococcus epidermidis were used as target genes for LAMP reaction.LAMP Primer design software Primer Explorer V5 was used to design multiple sets of LAMP primers for screening according to the highly conserved sequence of target genes.The primers of nuc-1,fecA-2 and SesB-4 with the best amplification effect were selected for subsequent experiments.2.By optimizing LAMP reaction system(Mg2+,Betaine,dNTPs concentration)and reaction conditions(temperature,time),The LAMP detection systems for Staphylococcus aureus,Escherichia coli and Staphylococcus epidermidis were successfully established.Then,with calcein as the color indicator,a visual LAMP detection method were constructed,and the detection results could be judged by color changes.Subsequently,Staphylococcus aureus,Escherichia coli and Staphylococcus epidermidis were used as positive controls respectively,and a dozen other common strains were used as negative controls for specificity verification.The results showed that the established LAMP method for three pathogenic bacteria only specifically recognized the target strain and the other strains were not amplified The results indicated that the established LAMP detection method has great specificity.Furthermore,the standard plasmid concentration of target strain was quantified by microspectrophotometer,and diluted ten-fold series dilutions to perform LAMP and PCR detection respectively.The results showed that the minimum detectable level of the established LAMP method for Staphylococcus aureus was 2.7×101 copies/μL,and the minimum detectable level of the established LAMP method for Escherichia coli and Staphylococcus epidermis were 5×101 copies/μL.The LAMP assay was 100-fold more sensitive than conventional PCR.3.The established LAMP detection methods for Staphylococcus aureus,Escherichia coli and Staphylococcus epidermidis were used to detect 210 clinical samples of dairy cow milk samples.The LAMP detection results were compared and analyzed with PCR.The results showed that the detection rate of Staphylococcus aureus by LAMP method was 29.5%,and the detection rate by conventional PCR method was 23.3%;The detection rate of Escherichia coli by LAMP method was 33.3%,and the detection rate of PCR method was 20.5%;The detection rate of Staphylococcus epidermidis by LAMP method was 12.4%,and that by conventional PCR method was 11%.It shows that the detection rate of the LAMP method were higher than conventional PCR.Conclusion:In this study,the rapid LAMP detection method for Staphylococcus aureus,Escherichia coli,and Staphylococcus epidermidis were established.The method is simple,rapid,high sensitivity,low-cost and the results are visualization.The LAMP assay can be used for the rapid detection of Staphylococcus aureus,Escherichia coli and Staphylococcus epidermidis in the field,which provide technical support for the rapid detection of the pathogenic bacteria of dairy cow mastitis.