改良LAMP检测鸡肉中单增李斯特氏菌的研究
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摘要
本研究建立一种改良环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术检测鸡肉中单增李斯特氏菌(Listeria monocytogenes,L. monocytogenes)的方法。针对单增李斯特氏菌的溶血素基因(hlyA)设计LAMP引物,对单增李斯特氏菌进行特异性检测,通过荧光曲线和肉眼观察荧光颜色来判定检测结果。试验结果表明:改良LAMP方法检测单增李斯特氏菌具有良好的特异性,7株单增李斯特氏菌呈阳性结果,24株非单增李斯特氏菌呈阴性结果。与聚合酶链反应(polymerase chain reaction,PCR)方法相比,改良LAMP方法具有灵敏度高(5.4×100 fg/μL)、检出限低(3.9×100 CFU/g)的特点,均是PCR结果的100倍。对66份鸡肉样品进行检测,得到改良LAMP方法的敏感性为100%,特异性为98.41%,符合率为98.48%。综上所述,改良LAMP方法能够快速、准确的检测单增李斯特氏菌,具有很好的应用前景。
In order to detect Listeria monocytogenes(L. monocytogenes),an improved loop-mediated isothermal amplification(improved LAMP)detection method was established. The hemolysin(hlyA)gene of L.monocytogenes was used to design the primers of improved LAMP and the detection results were shown by fluorescence curve or the fluorescence color by naked eye. 7 strains of L. monocytogenes were positive results and 24 strains of non-L. monocytogenes were negative results. The results indicated that the improved LAMP had a good specificity. Compared with Polymerase chain reaction(PCR),the improved LAMP method had a high sensitivity(5.4×100 fg/μL)and a low detection limits(3.9×100 CFU/g),which were both 100-folds of the results of PCR. The sensitivity,specificity and the coincidence rate of the improved LAMP method were 100 %,98.41 % and 98.48 %,respectively,by detecting 66 chicken samples. In conclusion,it is rapid and accurate for the detection of L. monocytogenes using improved LAMP method which has a good application prospect.
In order to detect Listeria monocytogenes(L. monocytogenes),an improved loop-mediated isothermal amplification(improved LAMP)detection method was established. The hemolysin(hlyA)gene of L.monocytogenes was used to design the primers of improved LAMP and the detection results were shown by fluorescence curve or the fluorescence color by naked eye. 7 strains of L. monocytogenes were positive results and 24 strains of non-L. monocytogenes were negative results. The results indicated that the improved LAMP had a good specificity. Compared with Polymerase chain reaction(PCR),the improved LAMP method had a high sensitivity(5.4×100 fg/μL)and a low detection limits(3.9×100 CFU/g),which were both 100-folds of the results of PCR. The sensitivity,specificity and the coincidence rate of the improved LAMP method were 100 %,98.41 % and 98.48 %,respectively,by detecting 66 chicken samples. In conclusion,it is rapid and accurate for the detection of L. monocytogenes using improved LAMP method which has a good application prospect.
引文
[1]张辉,王兴龙.食品中单核细胞增生性李斯特氏菌PCR快速检测[J].食品科学, 2008, 29(4):324-327
[2]刘海泉,赵强,孙晓红,等.多重PCR快速检测食品中的单核细胞增生性李斯特菌[J].中国农业科学, 2010, 43(23):4893-4900
[3] Lourenco A, Kamnetz M B, Gadotti C, et al. Antimicrobial treatments to control Listeria monocytogenes in queso fresco[J]. Food Microbiology, 2017, 64:47-55
[4] Song W J, Kang D H. Influence of water activity on inactivation of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in peanut butter by microwave heating[J]. Food Microbiology, 2016, 60:104-111
[5] Calvo T, Alvarez-Ordonez A, Prieto M, et al. Influence of processing parameters and stress adaptation on the inactivation of Listeria monocytogenes by Non-Thermal Atmospheric Plasma(NTAP)[J].Food Research International, 2016, 89(Pt 1):631-637
[6] Wang X, Uyttendaele M, Geeraerd A, et al. Thermal inactivation kinetics of surface contaminating Listeria monocytogenes on vacuumpackaged agar surface and ready-to-eat sliced ham and sausage[J].Food Research International, 2016, 89(Pt 1):843-849
[7] Du X J, Zhang X, Wang X Y, et al. Isolation and characterization of Listeria monocytogenes in Chinese food obtained from the central area of China[J]. Food Control, 2017, 74:9-16
[8] Jesus A L T, Fernandes M S, Kamimura B A, et al. Growth potential of Listeria monocytogenes in probiotic cottage cheese formulations with reduced sodium content[J]. Food Research International, 2016,81:180-187
[9] Wang Y, Li H, Luo L, et al. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogene[J]. International Journal Nanomedicine, 2017, 12:473-486
[10] Li F, Li B, Dang H, et al. Viable pathogens detection in fresh vegetables by quadruplex PCR[J]. LWT-Food Science and Technology,2017, 81:306-313
[11] V覿limaa A L, Tilsala T A, Virtanen E. Rapid detection and identification methods for Listeria monocytogenes in the food chain-a review[J]. Food Control, 2015, 55:103-114
[12] Day J B, Basavanna U. Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment[J]. Journal of Applied Microbiology, 2015,118(1):233-244
[13]刘芳,徐振娜,洪伟彬,等.食品中单增李斯特菌荧光PCR检测方法的建立[J].农村经济与科技, 2017(17):84-85
[14] Tang M J, Zhou S, Zhang X Y, et al. Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification[J]. Current Microbiology, 2011, 63(6):511-516
[15]顾思宇,张红星,金君华,等. LAMP法检测单核细胞增生性李斯特菌的研究[J].食品科技, 2016(8):297-301
[16] Liu H, Zhan F, Liu F, et al. Visual and sensitive detection of viable pathogenic bacteria by sensing of RNA markers in gold nanoparticles based paper platform[J]. Biosens Bioelectron, 2014, 62:38-46
[17] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):E63
[18] Yang Q, Wang F, Jones K L, et al. Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce[J]. Food Microbiology, 2015, 46:485-493
[19] Hongwarittorrn I, Chaichanawongsaroj N, Laiwattanapaisal W. Semi-quantitative visual detection of loop mediated isothermal amplification(LAMP)-generated DNA by distance-based measurement on a paper device[J]. Talanta, 2017, 175:135-142
[20]张体银,郑晶,黄晓蓉,等.环介导等温扩增技术快速检测食品中的单增李斯特氏菌[J].中国食品学报, 2010, 10(3):200-204
[21]洪伟鸣,宋亮,左伟勇.单增李斯特菌Hly基因的原核表达及多克隆抗体的制备[J].江西农业大学学报, 2017, 39(1):175-181
[22]张德福,赵禹宗,付绪磊,等.添加扩增内标的单增李斯特菌PCR检测方法的建立与应用[J].食品与发酵工业, 2016, 42(9):192-196
[23]李秀梅,梁智选,李颖,等.环介导等温扩增技术与横向流动试纸条法快速检测单增李斯特菌的研究[J].中国预防兽医学报,2016, 38(10):804-808
[24]杨粤,张蕴哲,张先舟,等.改良LAMP快速检测酸土脂环酸芽胞杆菌的研究[J].食品研究与开发, 2017, 38(10):165-172
[25]刘道亮,胡连霞,赵占民,等.改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157:H7[J].微生物学通报, 2011,38(3):430-435
[2]刘海泉,赵强,孙晓红,等.多重PCR快速检测食品中的单核细胞增生性李斯特菌[J].中国农业科学, 2010, 43(23):4893-4900
[3] Lourenco A, Kamnetz M B, Gadotti C, et al. Antimicrobial treatments to control Listeria monocytogenes in queso fresco[J]. Food Microbiology, 2017, 64:47-55
[4] Song W J, Kang D H. Influence of water activity on inactivation of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in peanut butter by microwave heating[J]. Food Microbiology, 2016, 60:104-111
[5] Calvo T, Alvarez-Ordonez A, Prieto M, et al. Influence of processing parameters and stress adaptation on the inactivation of Listeria monocytogenes by Non-Thermal Atmospheric Plasma(NTAP)[J].Food Research International, 2016, 89(Pt 1):631-637
[6] Wang X, Uyttendaele M, Geeraerd A, et al. Thermal inactivation kinetics of surface contaminating Listeria monocytogenes on vacuumpackaged agar surface and ready-to-eat sliced ham and sausage[J].Food Research International, 2016, 89(Pt 1):843-849
[7] Du X J, Zhang X, Wang X Y, et al. Isolation and characterization of Listeria monocytogenes in Chinese food obtained from the central area of China[J]. Food Control, 2017, 74:9-16
[8] Jesus A L T, Fernandes M S, Kamimura B A, et al. Growth potential of Listeria monocytogenes in probiotic cottage cheese formulations with reduced sodium content[J]. Food Research International, 2016,81:180-187
[9] Wang Y, Li H, Luo L, et al. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogene[J]. International Journal Nanomedicine, 2017, 12:473-486
[10] Li F, Li B, Dang H, et al. Viable pathogens detection in fresh vegetables by quadruplex PCR[J]. LWT-Food Science and Technology,2017, 81:306-313
[11] V覿limaa A L, Tilsala T A, Virtanen E. Rapid detection and identification methods for Listeria monocytogenes in the food chain-a review[J]. Food Control, 2015, 55:103-114
[12] Day J B, Basavanna U. Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment[J]. Journal of Applied Microbiology, 2015,118(1):233-244
[13]刘芳,徐振娜,洪伟彬,等.食品中单增李斯特菌荧光PCR检测方法的建立[J].农村经济与科技, 2017(17):84-85
[14] Tang M J, Zhou S, Zhang X Y, et al. Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification[J]. Current Microbiology, 2011, 63(6):511-516
[15]顾思宇,张红星,金君华,等. LAMP法检测单核细胞增生性李斯特菌的研究[J].食品科技, 2016(8):297-301
[16] Liu H, Zhan F, Liu F, et al. Visual and sensitive detection of viable pathogenic bacteria by sensing of RNA markers in gold nanoparticles based paper platform[J]. Biosens Bioelectron, 2014, 62:38-46
[17] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):E63
[18] Yang Q, Wang F, Jones K L, et al. Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce[J]. Food Microbiology, 2015, 46:485-493
[19] Hongwarittorrn I, Chaichanawongsaroj N, Laiwattanapaisal W. Semi-quantitative visual detection of loop mediated isothermal amplification(LAMP)-generated DNA by distance-based measurement on a paper device[J]. Talanta, 2017, 175:135-142
[20]张体银,郑晶,黄晓蓉,等.环介导等温扩增技术快速检测食品中的单增李斯特氏菌[J].中国食品学报, 2010, 10(3):200-204
[21]洪伟鸣,宋亮,左伟勇.单增李斯特菌Hly基因的原核表达及多克隆抗体的制备[J].江西农业大学学报, 2017, 39(1):175-181
[22]张德福,赵禹宗,付绪磊,等.添加扩增内标的单增李斯特菌PCR检测方法的建立与应用[J].食品与发酵工业, 2016, 42(9):192-196
[23]李秀梅,梁智选,李颖,等.环介导等温扩增技术与横向流动试纸条法快速检测单增李斯特菌的研究[J].中国预防兽医学报,2016, 38(10):804-808
[24]杨粤,张蕴哲,张先舟,等.改良LAMP快速检测酸土脂环酸芽胞杆菌的研究[J].食品研究与开发, 2017, 38(10):165-172
[25]刘道亮,胡连霞,赵占民,等.改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157:H7[J].微生物学通报, 2011,38(3):430-435