重组酶介导扩增方法快速检测A族乙型溶血性链球菌
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摘要
目的利用重组酶介导核酸扩增技术(RAA)建立快速检测A族乙型溶血性链球菌的方法。方法根据A族乙型溶血性链球菌细胞包膜蛋白酶A(Cep A)基因的保守序列设计引物和探针,通过构建含有目的基因片段的质粒分析方法的灵敏度,通过检测沙门菌、大肠杆菌、志贺菌和金黄色葡萄球菌标准菌株分析方法的特异性。结果建立的RAA方法在39℃,短时间内(<20 min)完成检测,灵敏度为10拷贝/μl,与沙门菌、大肠杆菌、志贺菌和金黄色葡萄球菌标准菌株无交叉反应,具有良好的特异性。结论建立的RAA方法速度快、特异性强、灵敏度高,适用于A族乙型溶血性链球菌的快速检测。
Objective To establish Recombinase aided amplification(RAA) assay for the detection of group A streptococcus pyogens.Methods A pair of primers and probe were designed based on Cep A gene sequence of group A streptococcus pyogens.The Cep A gene was cloned into a plasmid for sensitivity detection.The strains of Salmonella,Escherichia coli,Shigella and Staphylococcus aureus were used to test specificity.Results This RAA method was performed in a short time(<20 min) under the constant temperature of 39℃,with low detection limitation(10 copies/μl).This method had no cross reaction with Salmonella,Escherichia coli,Shigella and Staphylococcus aureus strains.Conclusion The RAA method was rapid,specific and sensitive.It could be used to detect group A streptococcus pyogens rapidly.
Objective To establish Recombinase aided amplification(RAA) assay for the detection of group A streptococcus pyogens.Methods A pair of primers and probe were designed based on Cep A gene sequence of group A streptococcus pyogens.The Cep A gene was cloned into a plasmid for sensitivity detection.The strains of Salmonella,Escherichia coli,Shigella and Staphylococcus aureus were used to test specificity.Results This RAA method was performed in a short time(<20 min) under the constant temperature of 39℃,with low detection limitation(10 copies/μl).This method had no cross reaction with Salmonella,Escherichia coli,Shigella and Staphylococcus aureus strains.Conclusion The RAA method was rapid,specific and sensitive.It could be used to detect group A streptococcus pyogens rapidly.
引文
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[14]郑伟,徐琦,罗鹏等.重组酶介导扩增方法快速检测黄热病毒[J].中国卫生检验杂志,2016,26(1):1-3.
[15]郑伟,王刚,杨永耀等.重组酶介导恒温扩增技术检测疟原虫方法的建立[J].寄生虫与医学昆虫学报,2016,23(1):15-20.
[16]Zhang XP,Guo LC,Ma RR,et al.Rapid detection of Salmonella with Recombinase Aided Amplification[J].Journal of Microbiological Methods,2017.DOI:10.1016/j.mimet.2017.06.011.
[17]张小平,郑乐怡,魏莹,等.重组酶介导扩增技术快速检测沙门菌方法的建立[J].中国国境卫生检疫杂志,2017,40(5):317-319.
[18]Carapetis JR,Steer AC,Mulholland EK,et al.The global burden of group A streptococcal diseases[J].Lancet Infectious Diseases,2005,5(11):685-694.
[2]梁云梅,杨永弘.A族β溶血性链球菌与猩红热的相关研究[J].微生物学杂志,2009,29(4):89-92.
[3]张一举.A组β型溶血性链球菌性咽炎的研究进展[J].新医学,2006,37(12):830-831.
[4]Schroeder BM.Diagnosis and management of group A streptococcal pharyngitis[J].American Family Physician,2003,67(4):880-883.
[5]Pedreanez A,Viera N,Rincon J,et al.Increased IL-6 in supernatant of rat mesangial cell cultures treated with erythrogenic toxin type B and its precursor isolated from nephritogenic streptococci[J].American Journal of Nephrology,2006,26(1):75-81.
[6]Viera N,Pedreanez A,Rincon J,et al.Streptococcal exotoxin Bincreases interleukin-6,tumor necrosis factor alpha,interleukin-8 and transforming growth factor beta-1 in leukocytes[J].Pediatric Nephrology,2007,22(9):1273-1281.
[7]Luo YH,Kuo CF,Huang KJ,et al.Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis[J].Kidney International,2007,72(6):716.
[8]Fitzmaurice J,Duffy G,Kilbride B,et al.Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef[J].Journal of Microbiological Methods,2004,59(2):243.
[9]O'Grady J,Ruttledge M,Sedanobalbás S,et al.Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR[J].Food Microbiology,2009,26(1):4-7.
[10]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[11]张也,刘以祥.美联免疫技术与食品安全快速检测[J].食品科学,2003,24(8):200-204.
[12]陈昱,潘迎捷,赵勇,等.基因芯片技术检测3种食源性致病微生物方法的建立[J].微生物学通报,2009,36(2):285-291.
[13]Carapetis JR,Steer AC,Mulholland EK,et al.The global burden of group A streptococcal diseases[J].Lancet Infectious Diseases,2005,5(11):685-694.
[14]郑伟,徐琦,罗鹏等.重组酶介导扩增方法快速检测黄热病毒[J].中国卫生检验杂志,2016,26(1):1-3.
[15]郑伟,王刚,杨永耀等.重组酶介导恒温扩增技术检测疟原虫方法的建立[J].寄生虫与医学昆虫学报,2016,23(1):15-20.
[16]Zhang XP,Guo LC,Ma RR,et al.Rapid detection of Salmonella with Recombinase Aided Amplification[J].Journal of Microbiological Methods,2017.DOI:10.1016/j.mimet.2017.06.011.
[17]张小平,郑乐怡,魏莹,等.重组酶介导扩增技术快速检测沙门菌方法的建立[J].中国国境卫生检疫杂志,2017,40(5):317-319.
[18]Carapetis JR,Steer AC,Mulholland EK,et al.The global burden of group A streptococcal diseases[J].Lancet Infectious Diseases,2005,5(11):685-694.