Establishment And Preliminary Application Of A Multiple PCR Method For Simultaneously Detecting Six Resistance Genes Of Streptococcus Suis

Streptococcus suis is an important pathogen of zoonotic pathogen,which can cause meningitis,endocarditis,pneumonia,arthritis,septicemia and other symptoms between humans and pigs after infection,not only causing serious economic damage to the development of pig industry,but also great harm to public health safety.Antibiotics are commonly used in the treatment of Streptococcus suis,but the unreasonable use of antibiotics has led to an increasingly serious problem of resistance in Streptococcus suis.At present,the commonly used detection methods for antibiotic resistance of isolated strains of Streptococcus suis in clinical practice are antimicrobial susceptibility test or single gene PCR detection methods,which are simple in operation but low in efficiency.In order to improve the efficiency of bacterial resistance testing,grasp the resistance situation of Streptococcus suis in China,and provide clinical antibiotic reference,this study established a six-fold multiplex PCR detection method for the resistance genes of Streptococcus suis.The specific research content is as follows:1.Preliminary establishment of a six-fold multiplex PCR detection method for resistance genes of Streptococcus suisBased on the prevalence of various resistance genes at home and abroad,as well as the results of resistance gene testing for isolated strains,a six-fold multiplex PCR detection method was established for the target genes of macrolide resistance gene erm B,tetracycline resistance gene tet(O),fluoroquinolone resistance gene pat B,aminoglycoside resistance gene aac(6″)-Ie-aph(2′)-Ia,oxazolidinone resistance gene optr A,and pleuromutilin resistance gene lsa(E).Using Primer 5.0 to design specific primers for gene conservation regions and establish single gene PCR detection methods Using Primer 5.0 to design specific primers for gene conservation regions and establish single gene PCR detection methods.A six-fold multiplex PCR detection method for resistance genes of Streptococcus suis was successfully established by exploring conditions such as optimal primer concentration,optimal annealing temperature,and optimal number of cycles;And the specificity,sensitivity,and repeatability of this method were evaluated in the laboratory.2.Comparative experiment of six-fold multiplex PCR detection method for resistance genes of Streptococcus suisComparison between the six-fold multiplex PCR detection method and the single gene PCR detection method: 12 strains of Streptococcus suis were tested for resistance genes using the six-fold multiplex PCR detection method and the single gene PCR detection method,respectively.The results showed that the coincidence rate of the two methods was 100%.Comparison of the results of the six-fold multiplex PCR detection method and the micro broth dilution method for antimicrobial susceptibility testing: The micro broth dilution method was used to determine the minimum inhibitory concentration(MIC)of various antibacterial antibiotic against Streptococcus suis.The comparison was made with the six-fold multiplex PCR detection method,and the statistical results showed that the coincidence rate of the two methods for the detection results of macrolides,tetracyclines,and oxazolidinones was 100%,The coincidence rate between the detection results of pleuromutilin and fluoroquinolones was 91.67%.3.Preliminary application of six-fold multiplex PCR detection method for Streptococcus suis resistance genesThe six-fold multiplex PCR detection method established in this study and micro broth dilution method were used to detect antibiotic resistance of 62 clinical isolates of Streptococcus suis.The results showed that the isolated strains had the strongest resistance to tylosin and azithromycin,with a resistance rate of 96.77%;The resistance rate to doxycycline and minocycline was 72.58%;The resistance rate to flufenicol was54.84%;The resistance rates to amikacin and gentamicin were 51.61% and 45.16%,respectively;The resistance rates to norfloxacin and enrofloxacin were 43.55% and33.87%,respectively;It is relatively sensitive to tiamulin,with a resistance rate of24.19%.Through further analysis of the carrying status of antibiotic resistance genes and the results of drug sensitivity tests,the results showed that the multiple resistance of the isolated strains reached 91.94%;Most of the strains showed three-fold resistance(46.77%)and four-fold resistance(38.71%),with the proportion of strains with five-fold and six-fold resistance being 4.84% and 1.61%,respectively.