Molecular Characteristics Analysis And Establishment Of MIRA-qPCR Detection Method For Staphylococcus

Objective Explore the pathogenic and genomic characteristics of Staphylococcus and establish a rapid,sensitive,and reliable MIRA-qPCR（Multienzyme isothermal rapid amplification combined with real-time quantitative PCR）detection method for Staphylococcus.Methods1.Staphylococcus from clinical sources was collected and its etiological characteristics were analyzed.MALDI-TOF MS mass spectrometry and 16S rRNA gene sequencing were used for species identification.The antibiotic sensitivity was detected by microbroth dilution method.2.The genome sequences of 57 Staphylococcus type strains were downloaded from the National Center for Biotechnology Information（NCBI）database and Quast v5.0.2 software was used for quality control of the genomes.Combined with RAST tool and BPGA v1.3 software,gene annotation and pan-genome analysis of genome sequences were performed.Virulence Factors of Pathogenic Bacteria（VFDB）and Comprehensive Antibiotic Resistance Database（CARD）were used to predict the distribution of virulence factors and drug resistance genes among different Staphylococcus,and the pheatmap package of R software was used to draw heat maps.3.The Oligo 7 software was used to design specific primers and probes,and a duplex MIRA-qPCR assay was established to detect Staphylococcus aureus and non-Staphylococcus aureus simultaneously.The nucleic acids of 28 strains of Staphylococcus and 33 strains of non-Staphylococcus were amplified by MIRA-qPCR to evaluate the specificity of the method.The nucleic acids of 10-fold gradient dilution of Staphylococcus aureus ATCC 12600^Twere amplified by MIRA-qPCR to evaluate the sensitivity of the method.The dried tofu,pork,milk and feces was artificially contaminated with bacteria suspension of 8 Staphylococcus strains to create simulated samples for MIRA-qPCR to evaluate the applicability.Results1.558 strains of Staphylococcus from clinical sources covering 22 species were collected,mainly S.aureus（274,49%）.The strains were mainly isolated from male samples（341,61.11%）,and most cases were aged from 61-80 years old（215,38.53%）.The main manifestations of staphylococcal positive cases were pneumonia,respiratory tract infection,suppurative infection and food poisoning.Strains were isolated from a wide range of sources,mainly from 19 samples such as secretions,blood,sputum,and urine.Strains had significant drug resistance,which were resistant to 23 kinds of antibiotics including penicillin,erythromycin,benzoxicillin and etc.And multi-drug resistance was dominant（494,88.53%）.2.Pan-genomic analysis showed that a total of 906 core genes,77709 accessory genes,7335 specific genes and 90 deletion genes were identified in 57Staphylococcus type strains.The functions of the genome can be divided into four categories including information storage and processing,metabolism,cell processing and signal transduction,and unknown characteristics.The metabolic pathways involved are mainly divided into six categories including cellular processes,environmental information processing,genetic information processing,human diseases,metabolism,and biological systems.The phylogenetic tree constructed based on the core genome of Staphylococcus formed nine clear evolutionary clusters,and the strains in the same evolutionary branch showed closer genetic relationships.57 strains of Staphylococcus carried 142 virulence factors.Toxin,Adherence and enzyme are the main potential virulence factors.A total of 46 drug-resistant genes were detected,the carrying rate of sdr M and sep A drug-resistant genes is over 90%.3.Three pairs specific primers of S.aureus species and Staphylococcus spp.were designed respectively,F₂R₁of S.aureus and F₃R₁of Staphylococcus spp.were selected as the most specific primers by conventional PCR.Based on the two pairs of specific primers and probes,a duplex MIRA-qPCR detection assay for S.aureus and other Staphylococcus was established.The detection method could complete nucleic acid amplification and detection after 20 min at 39℃,which showed strong specificity,high detection efficiency,high sensitivity and good repeatability.At the same time,it also demonstrated good practicability in simulated sample detection.Conclusion The etiological and genomic characteristics of Staphylococcus were analyzed,and a rapid,sensitive and reliable duplex MIRA-qPCR detection assay was established,which revealed the pathogenic characteristics of Staphylococcus from clinical sources,enriched the research of Staphylococcus genomics in China and provided a new choice for identifying Staphylococcus.