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范宏英, 龙敏, 刘欢, 罗军, 龙北国. 阪崎肠杆菌环介导等温扩增检测方法建立[J]. 中国公共卫生, 2011, 27(1): 31-33. DOI: 10.11847/zgggws2011-27-01-14
引用本文: 范宏英, 龙敏, 刘欢, 罗军, 龙北国. 阪崎肠杆菌环介导等温扩增检测方法建立[J]. 中国公共卫生, 2011, 27(1): 31-33. DOI: 10.11847/zgggws2011-27-01-14
FAN Hong-ying, LONG Min, LIU Huan, . Development of loop-mediated isothermal amplification(LAMP) method for detection of Enterobacter sakazakii[J]. Chinese Journal of Public Health, 2011, 27(1): 31-33. DOI: 10.11847/zgggws2011-27-01-14
Citation: FAN Hong-ying, LONG Min, LIU Huan, . Development of loop-mediated isothermal amplification(LAMP) method for detection of Enterobacter sakazakii[J]. Chinese Journal of Public Health, 2011, 27(1): 31-33. DOI: 10.11847/zgggws2011-27-01-14

阪崎肠杆菌环介导等温扩增检测方法建立

Development of loop-mediated isothermal amplification(LAMP) method for detection of Enterobacter sakazakii

  • 摘要: 目的 针对当前国内外传统检测方法缺点,建立阪崎肠杆菌快速检测法.方法 根据阪崎肠杆菌外膜蛋白(OmpA)基因,设计4条特异性引物(内、外引物各2条),建立并优化环介导等温扩增(loop-mediated isotherm alamp lification,LAMP)检测体系及反应条件,并进行LAMP反应的灵敏度、特异性实验及实际样品检测.结果 LAMP检测阪崎肠杆菌,优化后的条件选择为:Mg2+浓度6 mmol/L,dNTP浓度0.6 mmol/L,甜菜碱浓度0.8 mol/L,外引物与内引物的浓度比1:4,扩增温度58℃60min;细菌纯培养检测灵敏度为101cfu/mL;对11种细菌共26株菌进行LAMP扩增,仅8株阪崎肠杆菌得到阳性扩增结果,证明引物具有很高的特异性;对实际样品进行增菌检测,采用试剂盒提取DNA,从样品处理到报告结果,耗时26 h;并采用传统检测方法验证,正确性为100%.结论 该方法检测阪岐肠杆菌特异性强、灵敏度高,并且操作简便、检测成本低、耗时短,有望成为快速检测阪岐肠杆菌的有效方法.

     

    Abstract: Objective To establish a LAMP method for detection of Enterobacter sakazakii.Methods The loop-mediated isothermal amplification(LAMP) amplifying DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of Enterobacter sakazakii.A set of four primers,two outer and two inner,was designed specifically to recognize the outer membrane protein A gene(OmpA) of Enterobacter sakazakii.The LAMP reaction mix was optimized.Results The most optimal reaction temperature and time of the LAM Passay for the OmpAgene were 58 and 60 minutes, respectively.Genomic DNAs from 26 bacterial strains including 8 Enterobacter sakazakii strains were amplified using LAM P, and no amplicon was observed in other bacterial strains.The detection lmiit of the LAM Passay was a round 101 colonies forming units for pure cultures.In addition, this method was applied to detect food samples.Conclusion The results suggest that detection of Enterobac ter saka zakii by LAMP is an effective and lowcost procedure with high specificity and sensitivity that requires no specialized equipment.The assay is expected to become a valuable tool for rapid detection and identification of Enterobacter sakazakii.

     

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