摘要
目的建立检测生殖支原体(M.genitalium,Mg)的环介导等温扩增方法。方法根据Gen Bank公布的Mg核苷酸序列,设计4条特异性引物,进行PCR反应,对内、外引物浓度,d NTPs和Mg SO4浓度及Bst DNA聚合酶用量等进行优化,建立Mg LAMP的检测方法并进行特异性和敏感性评价;对20份HIV感染者的DNA样本分别进行LAMP和常规PCR检测。结果内、外引物浓度分别为1.6和0.2μmol/L,d NTPs浓度为1.6 mmol/L,Mg SO4浓度为12 mmol/L,Bst DNA聚合酶用量为8 U时,LAMP反应效果较好;用该体系检测同源关系较近的穿通支原体(M.penetrans,Mpe)、梨支原体(M.pirum,Mpi)均呈阴性;对Mg的检测灵敏度可达10-7;20份HIV感染者用LAMP法及常规PCR法各检出Mg阳性标本5份,两法的检测结果一致。结论建立的Mg LAMP检测方法的特异性、灵敏度较好,成本低廉,适合临床应用。
Objective To establish a loop-mediated isothermal amplification （LAMP） assay for detection of Mycoplasma genitalium （M. genitalium, Mg）. Methods Four specific primers of Mg were designed based on the sequence of Mg gene available in GenBank, and PCR was performed. Then, the concentrations of inner and outer primers, dNTPs, MgSO4 and Bst DNA polymerase were opti- mized, and the specificity and sensitivity of the established LAMP method for the detection of Mg were evaluated. Next, 20 DNA sam- pies from HIV-infected persons were detected by the LAMP method and conventional PCR, respectively. Results The optimized reac- tion conditions of the established LAMP method were as follows ： 1.6 ixmol/L of inner primer, 0.2 ixmol/L of out primer, 1.6 mmol/L of dNTPs, 12 mmol/L of MgSO4, and 8 U of Bst DNA polymerase. When the LAMP method was used to detect the DNA samples con- tained Mycoplasma penetrans or Mycoplasma pirum, the results were negative. The sensitivity of the method was 10-7 for the detection of Mg. When 20 DNA samples were detected by the LAMP method and conventional PCR, respectively, coincident results were ob- tained between them, both with 5 samples positive for Mg. Conclusion The established LAMP method with good specificity and sen- sitivity, and low cost, is suitable for the clinical detection of Mg.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2014年第11期860-862,共3页
Chinese Journal of Clinical Laboratory Science
基金
国家大学生创新训练计划项目(1210286101)
关键词
生殖支原体
环介导等温扩增
Mycoplasma genitalium
loop-mediated isothermal amplification
