食源性金黄色葡萄球菌肠毒素基因检测和基因分型研究
发布时间：2018-08-18 19:20
【摘要】：目的： 1建立食源性金黄色葡萄球菌14种肠毒素基因的多重PCR检测方法，调查石家庄地区食源性金葡菌中肠毒素基因的分布状况。 2设计食源性金葡菌的MLVA分型方法，比较MLST、MLVA、PFGE三种基因分型方法在食源性金葡菌基因多样性研究中的优劣势。 方法： 1对93株食源性金葡菌进行复苏传代培养，用试剂盒提取基因组DNA； 2设计14种肠毒素基因A、B、C、D、E、G、H、J、K、L、M、N、Q、U的PCR引物，通过优化反应条件确定多重PCR反应体系，对石家庄地区分离的93株食源性金葡菌进行肠毒素基因的检测； 3参考相关文献，结合网站数据设计金葡菌的MLVA分型方法。应用MLST、MLVA、PFGE三种方法分别对26株食源性金葡菌进行基因分型，对分型结果进行对比分析。 结果： 193株食源性金葡菌均成功复苏，提取的基因组DNA浓度和纯度均符合实验需求。 2设计的14种肠毒素基因PCR引物除SEE外，均扩增到了目的基因片段，双向测序结果在GeneBank上进行BLAST比对，证实为肠毒素基因的目的片段。 3设计优化的多重PCR反应体系能够成功扩增到食源性金葡菌的肠毒素基因目的片段，经检测，93株食源性金葡菌中79株检出含有肠毒素基因，肠毒素基因的检出率为84.95%；其中检出SEA的有55株（59.14%），SEG32株（34.41%），SEM32株（34.41%），SEK25株（26.88%），SEB22株（23.66%），SEU21株（22.58%），，SEN20株（21.51%），SEH16株（17.20%），SEJ15株（16.13%），SEC14株（15.05%），SED13株（13.98%），SEQ13株（13.98%），SEL7株（7.53%）；此外，57株金葡菌（61.29%）被检出同时含有两种或两种以上肠毒素基因，其中有1株检测到同时含有10种肠毒素基因，3株检测到同时含有9种，3株检测到同时含有8种。 4MLST分型将26株金葡菌分成8个ST序列型，其中ST59型11株，均来自同一起食物中毒分离获得的菌株；ST5型5株，ST464型3株，ST7型和ST15型各2株；此外还获得了一个新发现的ST序列型，已提交MLST国际数据库进行确认并收录。 5设计的MLVA方法具有较好的分型力和可重复性，26株食源性金葡菌经此方法成功分为10个基因型，其中VA05型11株，均为一起食物中毒中分离的菌株，VA07型4株，VA01型、VA09型和VA10型各两株，其他各一株。 6PFGE分型将26株金葡菌分为9组共13个基因型，其中A组共11株，均为一起食物中毒中分离的菌株，B组有4个亚型共4株，C组有两个亚型共3株，D组和E组各两株，其他组各一株。 结论： 1设计的14种肠毒素基因PCR引物以及多重PCR反应体系具有较好的稳定性，能够用于食源性金黄色葡萄球菌肠毒素基因的快速检测。 2MLVA的分型力较MLST强，但是不及PFGE。 3与MLST和PFGE相比，设计的MLVA分型方法具有快速简便、稳定性好、分型力较高等优点，可用于食源性金葡菌的快速基因分型。
[Abstract]:Objective: 1 to establish a multiplex PCR method for the detection of 14 enterotoxin genes of foodborne Staphylococcus aureus. To investigate the distribution of enterotoxin gene of Staphylococcus aureus in Shijiazhuang area. (2) to design the MLVA genotyping method of Staphylococcus aureus and compare the advantages and disadvantages of the three genotyping methods in the study of genetic diversity of Staphylococcus aureus. Methods: (1) 93 strains of Staphylococcus aureus were resuscitated and cultured, and genomic DNA was extracted by kit. (2) PCR primers were designed for 14 enterotoxin genes, the PCR primers were designed to determine the multiplex PCR reaction system by optimizing the reaction conditions. The enterotoxin genes of 93 strains of foodborne Staphylococcus aureus isolated in Shijiazhuang area were detected. (3) the MLVA typing method of Staphylococcus aureus was designed with reference to relevant literature and data from the website. The alleles of 26 strains of Staphylococcus aureus were genotyped by MLST-MLVAA-PFGE, and the results were compared and analyzed. Results: 193 strains of Staphylococcus aureus foodborne were successfully resuscitated, and the genomic DNA concentration and purity all met the experimental requirements. 2 the designed PCR primers of 14 enterotoxin genes were amplified with the exception of SEE. The results of bidirectional sequencing on GeneBank showed that it was the target fragment of enterotoxin gene. 3 the designed and optimized multiplex PCR reaction system could successfully amplify the target fragment of enterotoxin gene of Staphylococcus aureus. 缁忔娴
本文编号：2190434
[Abstract]:Objective: 1 to establish a multiplex PCR method for the detection of 14 enterotoxin genes of foodborne Staphylococcus aureus. To investigate the distribution of enterotoxin gene of Staphylococcus aureus in Shijiazhuang area. (2) to design the MLVA genotyping method of Staphylococcus aureus and compare the advantages and disadvantages of the three genotyping methods in the study of genetic diversity of Staphylococcus aureus. Methods: (1) 93 strains of Staphylococcus aureus were resuscitated and cultured, and genomic DNA was extracted by kit. (2) PCR primers were designed for 14 enterotoxin genes, the PCR primers were designed to determine the multiplex PCR reaction system by optimizing the reaction conditions. The enterotoxin genes of 93 strains of foodborne Staphylococcus aureus isolated in Shijiazhuang area were detected. (3) the MLVA typing method of Staphylococcus aureus was designed with reference to relevant literature and data from the website. The alleles of 26 strains of Staphylococcus aureus were genotyped by MLST-MLVAA-PFGE, and the results were compared and analyzed. Results: 193 strains of Staphylococcus aureus foodborne were successfully resuscitated, and the genomic DNA concentration and purity all met the experimental requirements. 2 the designed PCR primers of 14 enterotoxin genes were amplified with the exception of SEE. The results of bidirectional sequencing on GeneBank showed that it was the target fragment of enterotoxin gene. 3 the designed and optimized multiplex PCR reaction system could successfully amplify the target fragment of enterotoxin gene of Staphylococcus aureus. 缁忔娴
本文编号：2190434
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