乳酸菌饮料中嗜酸乳杆菌的实时荧光定量PCR检测方法
|
摘要
目的:为快速准确检测乳酸菌饮料中的嗜酸乳杆菌,建立实时荧光定量聚合酶链式反应(polymerase chainreaction,PCR)检测方法。方法:根据嗜酸乳杆菌NCFM的SPIDR保守区域设计特异性引物与探针,借助建立的实时荧光定量PCR方法进行特异性、灵敏度、重复性以及抗干扰能力验证,并利用模拟样品对方法进行检验,最后对市售的实际样品进行检测。结果:该方法的特异性较好;方法的绝对灵敏度达到3 pg,相对灵敏度达103 CFU/mL;重复性检测表明相对标准偏差在2.6%以下。同时进行杂菌干扰实验,在纯基因组水平和培养物水平混合大肠杆菌,扩增均无明显影响,表明建立的方法抗干扰能力较好。利用建立的实时荧光定量PCR方法对模拟样品进行检测并建立标准曲线,得出R~2为0.987,线性较好,可进行实际样品的检测。对市售的11份样品进行检测,其中6份含有嗜酸乳杆菌菌株,5份不含嗜酸乳杆菌菌株,标记嗜酸乳杆菌的样品全部检测出嗜酸乳杆菌且含量在5.83×10~2～3.68×1~04 CFU/mL之间。结论:建立的实时荧光定量PCR方法可快速、准确地检测出乳酸菌饮料中嗜酸乳杆菌。
Objective: To establish a real-time fluorescent quantitative PCR assay for the rapid and accurate detection of Lactobacillus acidophilus in probiotic beverages. Methods: Specific primers and probes were designed based on the conserved SPIDR region of L. acidophilus NCFM. The established method was assessed with respect to specificity,sensitivity and reproducibility, and it was applied to model and commercial samples. Results: The method had good speci?city. The absolute sensitivity was 3 pg and the relative sensitivity was 103 CFU/mL. The repeatability expressed as relative standard deviation(RSD) was below 2.6%. At the same time, the ampli?cation was not affected by the coexistence of pure genome or culture of Escherichia coli, indicating that the method has good anti-interference ability. This method showed good linearity(R~2 = 0.987) and it was feasible to detect actual samples. Of 11 commercial samples, 6 contained L. acidophilus strains while the rest did not. L. acidophilus was detected in all samples labeled with L. acidophilus in the range of 5.83 × 10~2 to 3.68 × 10~4 CFU/m L. Conclusion: The real-time ?uorescence quantitative PCR method can quickly and accurately detect L. acidophilus in probiotic beverages.
Objective: To establish a real-time fluorescent quantitative PCR assay for the rapid and accurate detection of Lactobacillus acidophilus in probiotic beverages. Methods: Specific primers and probes were designed based on the conserved SPIDR region of L. acidophilus NCFM. The established method was assessed with respect to specificity,sensitivity and reproducibility, and it was applied to model and commercial samples. Results: The method had good speci?city. The absolute sensitivity was 3 pg and the relative sensitivity was 103 CFU/mL. The repeatability expressed as relative standard deviation(RSD) was below 2.6%. At the same time, the ampli?cation was not affected by the coexistence of pure genome or culture of Escherichia coli, indicating that the method has good anti-interference ability. This method showed good linearity(R~2 = 0.987) and it was feasible to detect actual samples. Of 11 commercial samples, 6 contained L. acidophilus strains while the rest did not. L. acidophilus was detected in all samples labeled with L. acidophilus in the range of 5.83 × 10~2 to 3.68 × 10~4 CFU/m L. Conclusion: The real-time ?uorescence quantitative PCR method can quickly and accurately detect L. acidophilus in probiotic beverages.
引文
[1]梅林,王志耕.酸奶、乳酸菌饮料风味及其部分理化特性的研究[J].乳业科学与技术,2004,27(1):17-19.DOI:10.3969/j.issn.1671-5187.2004.01.003.
[2]郑坚强,司俊玲.改善嗜酸乳杆菌酸乳风味的研究[J].现代食品科技,2006,22(2):13-15.DOI:10.3969/j.issn.1673-9078.2006.02.004.
[3]BULATOVI?M L,KRUNIC T?,VUKA?INOVI?-SEKULI?M S,et al.Quality attributes of fermented whey-based beverage enriched with milk and probiotic strain[J].RSC Advances,2014,4(8):189-201.DOI:10.1039/C4RA08905G.
[4]ZHAO R,DUAN G,YANG T,et al.Purification,characterization and antibacterial mechanism of bacteriocin from L.acidophilus XH1[J].Tropical Journal of Pharmaceutical Research,2015,14(6):989-995.DOI:10.4314/tjpr.v14i6.8.
[5]杨天佑,段改丽,赵瑞香,等.嗜酸乳杆菌细菌素Lactobacillin XH1的生物学特性[J].食品科学,2013,34(17):197-200.DOI:10.7506/spkx1002-6630-201317042.
[6]AHMED Z,WANG Y P,CHENG Q L,et al.Lactobacillus acidophilus bacteriocin,from production to their application:an overview[J].African Journal of Biotechnology,2010,9(20):2843-2850.DOI:10.5897/AJB09.013.
[7]KUMAR A,ALREFAI W A,BORTHAKUR A,et al.Lactobacillus acidophilus counteracts enteropathogenic E.coli-induced inhibition of butyrate uptake in intestinal epithelial cells[J].American Journal of Physiology Gastrointestinal&Liver Physiology,2015,309(7):G602.DOI:10.1152/ajpgi.00186.2015.
[8]AFFHAN S,DACHANG W,XIN Y,et al.Lactic acid bacteria protect human intestinal epithelial cells from Staphylococcus aureus and Pseudomonas aeruginosa infections[J].Genetics&Molecular Research,2015,14(4):17044.DOI:10.4238/2015.December.16.5.
[9]REID G,BURTON J.Use of Lactobacillus to prevent infection by pathogenic bacteria[J].Microbes and Infection,2002,4(3):319-324.DOI:10.1016/s1286-4579(02)01544-7.
[10]YING H,ZHENG Y C.The probiotic Lactobacillus acidophilus reduces cholesterol absorption through the down-regulation of Niemann-Pick C1-like 1 in Caco-2 cells[J].British Journal of Nutrition,2010,103(4):473-478.DOI:10.1017/S0007114509991991.
[11]李杰,李炳仁,李琴.乳制品中嗜酸乳杆菌的益生特性[J].黑龙江八一农垦大学学报,2011,23(3):43-46.DOI:10.3969/j.issn.1002-2090.2011.03.012.
[12]郑红星,祁珊珊.嗜酸乳杆菌的研究进展[J].黑龙江畜牧兽医,2015(17):61-64.DOI:10.13881/j.cnki.hljxmsy.2015.1561.
[13]林芝,曹高芬,杨章民.守护肠道微生态系统的健康卫士:益生菌[J].中学生物教学,2017(18):71-72.
[14]GAREAU M G,SHERMAN P M,WALKER W A.Probiotics and the gut microbiota in intestinal health and disease[J].Nat Rev Gastroenterol Hepatol,2010,7(9):503-514.DOI:10.1038/nrgastro.2010.117.
[15]DONG-NEI H E,ZHU H M,LAI W D.Influence of active Lactobacillus beverage on intestinal flora of human[J].Chinese Journal of Microecology,2006,18(6):454-456.10.13381/j.cnki.cjm.2006.06.013.
[16]单尹佩,徐波.我国乳酸菌饮料产业化现状及发展策略[J].科技广场,2011(12):72-75.DOI:10.3969/j.issn.1671-4792.2011.12.018.
[17]卫生部.可用于食品的菌种名单:卫办监督发[2010]65号[S].2010.
[18]国家卫生和计划生育委员会.国家卫计委关于食品中使用菌种标签标示有关问题的复函:卫办监督函[2013]367号[S].2013.
[19]卫生部.国家标准预包装食品标签通则:GB 7718-2011[S].北京:中国标准出版社,2011.
[20]国家卫生和计划生育委员会.饮料:GB 7101-2015[S].北京:中国标准出版社,2015.
[21]国家卫生和计划生育委员会,国家食品药品监督管理总局.食品微生物学检验乳酸菌检验:GB 4789.35-2016[S].北京:中国标准出版社,2016.
[22]BUSTIN S A,BENES V,NOLAN T,et al.Quantitative real-time RT-PCR:a perspective[J].Journal of Molecular Endocrinology,2005,34(3):597-601.DOI:10.1677/jme.1.01755.
[23]陈旭,齐凤坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010(8):148-155.DOI:10.3969/j.issn.1005-9369.2010.08.029.
[24]郑维,权春善,朴永哲,等.一种快速提取细菌总D N A的方法研究[J].中国生物工程杂志,2006,26(4):75-80.DOI:10.3969/j.issn.1671-8135.2006.04.014.
[25]YAN-TAO W U,LIU X L,CAO Y,et al.Detection of bifidobacteria in fermented dairy products by real-time fluorescent quantitative PCR[J].Food Science,2013,34(8):172-175.DOI:10.7506/spkx1002-6630-201308036.
[26]程海星,郭月英,任霆,等.实时荧光定量P C R技术原理及在食品检测中的应用[J].食品与发酵工业,2015,41(3):243-247.DOI:10.13995/j.cnki.11-1802/ts.201503043.
[27]BINETTI A G,CAPRA M L,ALVAREZ M A,et al.PCRmethod for detection and identification of Lactobacillus casei/paracasei bacteriophages in dairy products[J].International Journal of Food Microbiology,2008,124(2):147-153.DOI:10.1016/j.ijfoodmicro.2008.03.006.
[28]谢雪钦.Taq Man实时荧光聚合酶链式反应(PCR)技术定量检测婴幼儿配方食品中的金黄色葡萄球菌[J].食品与发酵工业,2016,42(7):223-229.DOI:10.13995/j.cnki.11-1802/ts.201607037.
[29]GUO Z H,FANG H,XIA Z S,et al.Detection of Lactobacillus acidophilus in fermented material by real-time fluorescent quantitative PCR[J].Animal Husbandry&Feed Science,2016,1:54-57.DOI:10.19578/j.cnki.ahfs.2016.01.016.
[30]包秋华,李梅花,徐洁,等.一种检测发酵乳中嗜酸乳杆菌的定量定性方法[J].食品研究与开发,2012,33(11):153-155.
[31]SHEU S J,HWANG W Z,CHIANG Y C,et al.Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods[J].Journal of Food Science,2010,75(8):521-527.DOI:10.1111/j.1750-3841.2010.01816.x.
[32]FURET J P,QU N E P,TAILLIEZ P.Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR[J].International Journal of Food Microbiology,2004,97(2):197-207.DOI:10.1016/j.ijfoodmicro.2004.04.020.
[33]赵文静,李妍,高鹏飞,等.实时荧光定量PCR技术在乳酸菌定量检测中的应用[J].食品与生物技术学报,2009,28(4):433-437.DOI:10.3321/j.issn:1673-1689.2009.04.001.
[2]郑坚强,司俊玲.改善嗜酸乳杆菌酸乳风味的研究[J].现代食品科技,2006,22(2):13-15.DOI:10.3969/j.issn.1673-9078.2006.02.004.
[3]BULATOVI?M L,KRUNIC T?,VUKA?INOVI?-SEKULI?M S,et al.Quality attributes of fermented whey-based beverage enriched with milk and probiotic strain[J].RSC Advances,2014,4(8):189-201.DOI:10.1039/C4RA08905G.
[4]ZHAO R,DUAN G,YANG T,et al.Purification,characterization and antibacterial mechanism of bacteriocin from L.acidophilus XH1[J].Tropical Journal of Pharmaceutical Research,2015,14(6):989-995.DOI:10.4314/tjpr.v14i6.8.
[5]杨天佑,段改丽,赵瑞香,等.嗜酸乳杆菌细菌素Lactobacillin XH1的生物学特性[J].食品科学,2013,34(17):197-200.DOI:10.7506/spkx1002-6630-201317042.
[6]AHMED Z,WANG Y P,CHENG Q L,et al.Lactobacillus acidophilus bacteriocin,from production to their application:an overview[J].African Journal of Biotechnology,2010,9(20):2843-2850.DOI:10.5897/AJB09.013.
[7]KUMAR A,ALREFAI W A,BORTHAKUR A,et al.Lactobacillus acidophilus counteracts enteropathogenic E.coli-induced inhibition of butyrate uptake in intestinal epithelial cells[J].American Journal of Physiology Gastrointestinal&Liver Physiology,2015,309(7):G602.DOI:10.1152/ajpgi.00186.2015.
[8]AFFHAN S,DACHANG W,XIN Y,et al.Lactic acid bacteria protect human intestinal epithelial cells from Staphylococcus aureus and Pseudomonas aeruginosa infections[J].Genetics&Molecular Research,2015,14(4):17044.DOI:10.4238/2015.December.16.5.
[9]REID G,BURTON J.Use of Lactobacillus to prevent infection by pathogenic bacteria[J].Microbes and Infection,2002,4(3):319-324.DOI:10.1016/s1286-4579(02)01544-7.
[10]YING H,ZHENG Y C.The probiotic Lactobacillus acidophilus reduces cholesterol absorption through the down-regulation of Niemann-Pick C1-like 1 in Caco-2 cells[J].British Journal of Nutrition,2010,103(4):473-478.DOI:10.1017/S0007114509991991.
[11]李杰,李炳仁,李琴.乳制品中嗜酸乳杆菌的益生特性[J].黑龙江八一农垦大学学报,2011,23(3):43-46.DOI:10.3969/j.issn.1002-2090.2011.03.012.
[12]郑红星,祁珊珊.嗜酸乳杆菌的研究进展[J].黑龙江畜牧兽医,2015(17):61-64.DOI:10.13881/j.cnki.hljxmsy.2015.1561.
[13]林芝,曹高芬,杨章民.守护肠道微生态系统的健康卫士:益生菌[J].中学生物教学,2017(18):71-72.
[14]GAREAU M G,SHERMAN P M,WALKER W A.Probiotics and the gut microbiota in intestinal health and disease[J].Nat Rev Gastroenterol Hepatol,2010,7(9):503-514.DOI:10.1038/nrgastro.2010.117.
[15]DONG-NEI H E,ZHU H M,LAI W D.Influence of active Lactobacillus beverage on intestinal flora of human[J].Chinese Journal of Microecology,2006,18(6):454-456.10.13381/j.cnki.cjm.2006.06.013.
[16]单尹佩,徐波.我国乳酸菌饮料产业化现状及发展策略[J].科技广场,2011(12):72-75.DOI:10.3969/j.issn.1671-4792.2011.12.018.
[17]卫生部.可用于食品的菌种名单:卫办监督发[2010]65号[S].2010.
[18]国家卫生和计划生育委员会.国家卫计委关于食品中使用菌种标签标示有关问题的复函:卫办监督函[2013]367号[S].2013.
[19]卫生部.国家标准预包装食品标签通则:GB 7718-2011[S].北京:中国标准出版社,2011.
[20]国家卫生和计划生育委员会.饮料:GB 7101-2015[S].北京:中国标准出版社,2015.
[21]国家卫生和计划生育委员会,国家食品药品监督管理总局.食品微生物学检验乳酸菌检验:GB 4789.35-2016[S].北京:中国标准出版社,2016.
[22]BUSTIN S A,BENES V,NOLAN T,et al.Quantitative real-time RT-PCR:a perspective[J].Journal of Molecular Endocrinology,2005,34(3):597-601.DOI:10.1677/jme.1.01755.
[23]陈旭,齐凤坤,康立功,等.实时荧光定量PCR技术研究进展及其应用[J].东北农业大学学报,2010(8):148-155.DOI:10.3969/j.issn.1005-9369.2010.08.029.
[24]郑维,权春善,朴永哲,等.一种快速提取细菌总D N A的方法研究[J].中国生物工程杂志,2006,26(4):75-80.DOI:10.3969/j.issn.1671-8135.2006.04.014.
[25]YAN-TAO W U,LIU X L,CAO Y,et al.Detection of bifidobacteria in fermented dairy products by real-time fluorescent quantitative PCR[J].Food Science,2013,34(8):172-175.DOI:10.7506/spkx1002-6630-201308036.
[26]程海星,郭月英,任霆,等.实时荧光定量P C R技术原理及在食品检测中的应用[J].食品与发酵工业,2015,41(3):243-247.DOI:10.13995/j.cnki.11-1802/ts.201503043.
[27]BINETTI A G,CAPRA M L,ALVAREZ M A,et al.PCRmethod for detection and identification of Lactobacillus casei/paracasei bacteriophages in dairy products[J].International Journal of Food Microbiology,2008,124(2):147-153.DOI:10.1016/j.ijfoodmicro.2008.03.006.
[28]谢雪钦.Taq Man实时荧光聚合酶链式反应(PCR)技术定量检测婴幼儿配方食品中的金黄色葡萄球菌[J].食品与发酵工业,2016,42(7):223-229.DOI:10.13995/j.cnki.11-1802/ts.201607037.
[29]GUO Z H,FANG H,XIA Z S,et al.Detection of Lactobacillus acidophilus in fermented material by real-time fluorescent quantitative PCR[J].Animal Husbandry&Feed Science,2016,1:54-57.DOI:10.19578/j.cnki.ahfs.2016.01.016.
[30]包秋华,李梅花,徐洁,等.一种检测发酵乳中嗜酸乳杆菌的定量定性方法[J].食品研究与开发,2012,33(11):153-155.
[31]SHEU S J,HWANG W Z,CHIANG Y C,et al.Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods[J].Journal of Food Science,2010,75(8):521-527.DOI:10.1111/j.1750-3841.2010.01816.x.
[32]FURET J P,QU N E P,TAILLIEZ P.Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR[J].International Journal of Food Microbiology,2004,97(2):197-207.DOI:10.1016/j.ijfoodmicro.2004.04.020.
[33]赵文静,李妍,高鹏飞,等.实时荧光定量PCR技术在乳酸菌定量检测中的应用[J].食品与生物技术学报,2009,28(4):433-437.DOI:10.3321/j.issn:1673-1689.2009.04.001.