高效液相色谱法提高红曲细菌分离与分析的效果
|
摘要
为了探讨高效液相色谱技术(HPLC)在复杂微生态体系中微生物分离、分析的应用潜力,提高现代分子生物学技术检测的灵敏度和准确性,本文选取红曲作为模式体系,优化红曲中细菌样品的制备方法,应用HPLC技术对红曲中的生产菌株进行分离,并结合变性梯度凝胶电泳(DGGE)、细菌16S rDNA鉴定方法对色谱分离得到的菌株进行鉴定。结果显示:根据HPLC紫外检测图谱收集到5个明显的洗脱峰,其中有3个洗脱峰经DGGE分析为单一条带,16S rDNA鉴定洗脱峰分别包括环状芽孢杆菌、铜绿假单胞菌、人参土芽孢杆菌和脂肪酸芽孢杆菌。研究表明,高效液相色谱技术能够从复杂的红曲微生物样品中分离出单一的细菌,与传统PCRDGGE技术相比,无需进行胶回收便可实现16S rDNA的直接测序,且具有准确性高,试验周期短,细菌细胞可回收等优点。色谱分离技术在下一代测序技术中的应用潜力也在探讨中。
Objectives:To eveluate the potential application of HPLC(High Performance Liquid Chromatography) to the seperation and analysis of microbe in micro-ecosystem,so that improves the sensitivity and accuracy of the techniques of modern molecular biology.Methods:Red kojic rice was selected as a model system,bacteria inside was separated and recovered by the optimized sample preparation procedure and chromatographic conditions of HPLC,then analyzed by PCR-DGGE(Denaturing Gradient Gel Electrophoresis) and identificated by 16S rDNA sequencing.Results:5 fractions were recovered,and there were 3 fractions might be pure strains based on their electrophoretogram of PCR-DGGE.Direct sequencing of 16 S rDNA of these 3 fractions contained Bacillus circulans,Bacillus ginsengihumi,Pseudomonas sp.or Alicyclobacillus respectively.Conclusions:This study highlights the HPLC technique could be a rapid,accurate and recoverable method for the separation of pure strain from Red Kojic Rice.Compared with the traditional PCR-DGGE technique,it can realize the direct sequencing of longer sequence of 16 S rDNA without gel recovery.The potential application of the approach of chromatographic separation in the next generation sequencing is also discussed.
Objectives:To eveluate the potential application of HPLC(High Performance Liquid Chromatography) to the seperation and analysis of microbe in micro-ecosystem,so that improves the sensitivity and accuracy of the techniques of modern molecular biology.Methods:Red kojic rice was selected as a model system,bacteria inside was separated and recovered by the optimized sample preparation procedure and chromatographic conditions of HPLC,then analyzed by PCR-DGGE(Denaturing Gradient Gel Electrophoresis) and identificated by 16S rDNA sequencing.Results:5 fractions were recovered,and there were 3 fractions might be pure strains based on their electrophoretogram of PCR-DGGE.Direct sequencing of 16 S rDNA of these 3 fractions contained Bacillus circulans,Bacillus ginsengihumi,Pseudomonas sp.or Alicyclobacillus respectively.Conclusions:This study highlights the HPLC technique could be a rapid,accurate and recoverable method for the separation of pure strain from Red Kojic Rice.Compared with the traditional PCR-DGGE technique,it can realize the direct sequencing of longer sequence of 16 S rDNA without gel recovery.The potential application of the approach of chromatographic separation in the next generation sequencing is also discussed.
引文
[1]TORTORA G J,FUNKE B R,CASE C L.Microbiology[M].Redwood City,The Benjamin/Cummings Publishing Company,Inc.,CA,1992:174.
[2]聂志强,王敏,郑宇.3种分子生物学技术在传统发酵食品微生物多样性研究中的应用[J].食品科学,2012,33(23):346-350.
[3]ACHBERGLUND R,LINDBERG A A.Rapid and sensitive detection of Shigella sonnei in feces by the use of an O-antigen-specific monoclonal antibody in a combined immunomagnetic separation-polymerase chain reaction assay[J].Clinical Microbiology&Infection the Official Publication of the European Society of Clinical Microbiology&Infectious Diseases,1996,2(1):55-58.
[4]赵璐,苏战强,周银萍,等.免疫磁珠法分离大肠杆菌O157:H7及毒力基因的PCR检测[J].中国兽医杂志,2015,51(5):77-79.
[5]RESCHIGLIAN P,ZATTONI A,RODA B,et al Field-flow fractionation and biotechnology[J].Trends in Biotechnology,2005,23(9):475-483.
[6]LEFTVER S,VANDESONPELE J,SPELEMAN F,et al.RT Primer DB:the portal for real-time PCRprimers and probes[J].Nucleic Acids Research,2009,37(Database issue):942-945.
[7]KIM S E,CHOI S C,PARK K S,et al.Change of fecal flora and effectiveness of the short-term VSL#3 probiotic treatment in patients with functional constipation[J].Journal of Neurogastroenterology&Motility,2015,21(1):111-120.
[8]QUEIPOORTU譙O M I,SEOANE L M,MURRI M,et al.Gut microbiota composition in male rat models under different nutritional status and physical activity and its association with serum leptin and ghrelin levels[J].Plos One,2013,8(5):65465-65465.
[9]仪淑敏,王学琦,励建荣,等.鱼糜制品细菌菌群多样性的PCR-DGGE方法建立[J].中国食品学报,2014,14(7):192-197.
[10]吴高峰,李文刚,高卫科,等.PCR-DGGE的原理及在动物肠道菌群分析中的应用[J].中国畜牧兽医,2008,35(6):37-39.
[11]黄卫强,张和平.分子生物学技术在肠道菌群研究中的进展[J].微生物学通报,2014,6(41):1195-1202.
[12]STEFANIS C,MANTZOURANI I,PLESSAS S,et al.Reviewing classical and molecular techniques regarding profiling of probiotic character of microorganisms[J].Current Research in Nutrition and Food Science,2016,4(1):27-47.
[13]YANG C H,CROWLEY D E.Rhizosphere microbial community structure in relation to root location and plant iron nutritional status[J].Applied&Environmental Microbiology,2000,66(1):345-551.
[14]PLIEVA F M,GALAEV IYU,MATTIASSON BCryogel applications in microbiology[J].Trends Microbiol,2008,16(11):543-551.
[15]LIU S T,CHEN Z H,XIE J B,et al.High-performance ion exchange chromatography of intact bacterial cells in the manner of molecules:1.establishment of methodology[J].Anal Chem,2010,82(20):8544-8550.
[16]ZHENG X F,ZHU Y J,LIU B,et al.Rapid differentiation of Ralstoniia solanacearum avirulent and virulent strains by cell fraction of an isolate using high performance liquid chromatography[J].Microbial Pathogenesis,2016,90:84-92.
[17]LIN J,GAO Z N,LIU S T,et al.Adsorption characteristics of cell surface of ralstonia solanacearum wild type strain and avirulent mutant[J].Chinese Journal of Applied&Environmental Biology,2010,16(4):499-503.
[18]陈昭华,刘树滔,薛伟明,等.对数期金黄色葡萄球菌的离子交换色谱行为及其表征[J].色谱,2004,22(3):234-236.
[19]陈昭华,刘树滔,林娟,等.肠出血性大肠杆菌O157:H7的高效液相离子交换色谱特征分析[J].食品科学,2012,33(3):197-201.
[20]陈章捷,时祥柱,林娟,等.采用高效离子交换色谱分离分析动物肠道菌群方法初探[J].应用与环境生物学报,2006,12(2):278-282.
[2]聂志强,王敏,郑宇.3种分子生物学技术在传统发酵食品微生物多样性研究中的应用[J].食品科学,2012,33(23):346-350.
[3]ACHBERGLUND R,LINDBERG A A.Rapid and sensitive detection of Shigella sonnei in feces by the use of an O-antigen-specific monoclonal antibody in a combined immunomagnetic separation-polymerase chain reaction assay[J].Clinical Microbiology&Infection the Official Publication of the European Society of Clinical Microbiology&Infectious Diseases,1996,2(1):55-58.
[4]赵璐,苏战强,周银萍,等.免疫磁珠法分离大肠杆菌O157:H7及毒力基因的PCR检测[J].中国兽医杂志,2015,51(5):77-79.
[5]RESCHIGLIAN P,ZATTONI A,RODA B,et al Field-flow fractionation and biotechnology[J].Trends in Biotechnology,2005,23(9):475-483.
[6]LEFTVER S,VANDESONPELE J,SPELEMAN F,et al.RT Primer DB:the portal for real-time PCRprimers and probes[J].Nucleic Acids Research,2009,37(Database issue):942-945.
[7]KIM S E,CHOI S C,PARK K S,et al.Change of fecal flora and effectiveness of the short-term VSL#3 probiotic treatment in patients with functional constipation[J].Journal of Neurogastroenterology&Motility,2015,21(1):111-120.
[8]QUEIPOORTU譙O M I,SEOANE L M,MURRI M,et al.Gut microbiota composition in male rat models under different nutritional status and physical activity and its association with serum leptin and ghrelin levels[J].Plos One,2013,8(5):65465-65465.
[9]仪淑敏,王学琦,励建荣,等.鱼糜制品细菌菌群多样性的PCR-DGGE方法建立[J].中国食品学报,2014,14(7):192-197.
[10]吴高峰,李文刚,高卫科,等.PCR-DGGE的原理及在动物肠道菌群分析中的应用[J].中国畜牧兽医,2008,35(6):37-39.
[11]黄卫强,张和平.分子生物学技术在肠道菌群研究中的进展[J].微生物学通报,2014,6(41):1195-1202.
[12]STEFANIS C,MANTZOURANI I,PLESSAS S,et al.Reviewing classical and molecular techniques regarding profiling of probiotic character of microorganisms[J].Current Research in Nutrition and Food Science,2016,4(1):27-47.
[13]YANG C H,CROWLEY D E.Rhizosphere microbial community structure in relation to root location and plant iron nutritional status[J].Applied&Environmental Microbiology,2000,66(1):345-551.
[14]PLIEVA F M,GALAEV IYU,MATTIASSON BCryogel applications in microbiology[J].Trends Microbiol,2008,16(11):543-551.
[15]LIU S T,CHEN Z H,XIE J B,et al.High-performance ion exchange chromatography of intact bacterial cells in the manner of molecules:1.establishment of methodology[J].Anal Chem,2010,82(20):8544-8550.
[16]ZHENG X F,ZHU Y J,LIU B,et al.Rapid differentiation of Ralstoniia solanacearum avirulent and virulent strains by cell fraction of an isolate using high performance liquid chromatography[J].Microbial Pathogenesis,2016,90:84-92.
[17]LIN J,GAO Z N,LIU S T,et al.Adsorption characteristics of cell surface of ralstonia solanacearum wild type strain and avirulent mutant[J].Chinese Journal of Applied&Environmental Biology,2010,16(4):499-503.
[18]陈昭华,刘树滔,薛伟明,等.对数期金黄色葡萄球菌的离子交换色谱行为及其表征[J].色谱,2004,22(3):234-236.
[19]陈昭华,刘树滔,林娟,等.肠出血性大肠杆菌O157:H7的高效液相离子交换色谱特征分析[J].食品科学,2012,33(3):197-201.
[20]陈章捷,时祥柱,林娟,等.采用高效离子交换色谱分离分析动物肠道菌群方法初探[J].应用与环境生物学报,2006,12(2):278-282.