利用JP探针（Junction Probe）技术快速检测金黄色葡萄球菌
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摘要
目的：1.通过对基于限制性内切酶核酸等温检测技术的认识和研究,旨在建立一种适合实验室的,并可以广泛推广到基层实验医学实验室的JP(Junction Probe)探针技术。
2.应用JP探针技术实现金黄色葡萄球菌(Staphyloccocus aureus)的快速检测。
方法：1、设计三套JP探针体系,通过对合成的金黄色葡萄球菌特异性单链的荧光检测来筛选出酶切效率最高,反应稳定性最好的探针体系,同时优化模板探针反应比、探究探针检测特异性以及检测双链的特性。
2、在深入了解金黄色葡萄球菌生物学特性的基础上,对从医院获得的菌株,进行扩大培养并通过平板培养获得单克隆,作为后续的实验材料。
3、针对步骤1、2,结合PCR扩增技术以及磁珠提取单链技术在获得大量目标单链的基础上,实现JP探针对金黄色葡萄球菌的快速检测。
结果：1、应用JP探针检测金黄色葡萄球菌合成单链的结果表明,该方法检测金黄色葡萄球菌的可行性,建立了一种新的快速核酸检测方法。本实验通过设计三套不同的探针体系筛选出了PA1-PB1探针系统,其反应速率快,反应稳定性好,适合进行后续菌的检测；PA1-PB1探针体系优化的实验结果表明,该探针体系可以达到的检测下限是4nM,另外该探针体系可以很好的鉴别金黄色葡萄球菌以及与目标菌基因序列最相近的表皮葡萄球菌。同时合成目标单链与互补单链形成的双链探针反应,表明目前的探针体系还不能很好的检测双链,这就要求在检测临床菌时,必须制备出一定长度的单链才能有效进行。
2、通过对金黄色葡萄球菌的培养结果表明,本实验的研究菌对生长条件要求不高,在LB培养基上就可以生长良好,为后续的实验提供了大量稳定的实验材料。
3、应用JP探针检测金黄色葡萄球菌的实验表明,该方法能够快速而有效的鉴定金黄色葡萄,较传统的生化鉴定方法具有很大的优势。应用PCR扩增金黄色葡萄球菌,得到了大量的检测片段,达到第一级的放大；由于JP探针检测的目标片段是单链,为此本实验结合磁珠技术快速的提取得到了大量高纯度的单链。应用PA1-PB1探针体系检测磁珠提取单链,实现荧光信号的二级放大,从而达到仪器可检测的范围。通过以上两级放大本实验的阳性检出率达到97.25%,较单纯使用PCR技术更准确。
Purpose:1. To establish an isothermal nucleic acid detection method, Junction Probe (JP), based on restriction endonuclease, suitable for both research and clinic laboratory.
2. To develop a rapid method to detect Staphyloccocus aureus using JP technology.
Method:1. Three pairs of probes were designed and tested. By using the probes with highest reactivity and reaction reproducibility, the molar ratio of the probes was optimized, the detection specificity was investigated and the double-stranded targets were tested.
2. Based on the biological nature of Staphyloccocus aureus, the bacterial strains from hospitals were grown on LB agar and the genomic DNA were obtained by the purification of alkali treatment method.
3. Single strand DNA were obtained by combining PCR amplification and magnetic beads extraction, and Staphyloccocus aureus was detected using JP technology.
Discussion:1.The feasibility of the method to detect Staphyloccocus aureus was realized by detecting the artificial single-strand DNA target. The PA1-PB1probe pair was screened to give the fastest reaction rate and best reproducibility. The experiments showed that the detection limit of this system is4nM of temple DNA, and it can distinguish Staphylococcus aureus from Staphylococcus epidermidis, which contains SNP in the target sequence. The experiments also showed that the systems can not detect double stranded DNA, so single stranded target must be prepared while detecting clinical samples.
2.The cultivation results of Staphylococcus aureus showed that the bacteria does not need stringent growing conditions, and LB medium is good enough, which satisfied our subsequent experimental requirement.
3.This method can quickly and effectively detect Staphylococcus aureus, and has great advantages over traditional biochemical identification methods. The system has a great S/N ratio, resulting from both of the PCR and JP signal amplification in the detection of single-strand DNA samples. With the two-stage examination, the positive ratio is up to97.25%by our PCR-JP method, which is more specific than PCR technology alone.
2.应用JP探针技术实现金黄色葡萄球菌(Staphyloccocus aureus)的快速检测。
方法：1、设计三套JP探针体系,通过对合成的金黄色葡萄球菌特异性单链的荧光检测来筛选出酶切效率最高,反应稳定性最好的探针体系,同时优化模板探针反应比、探究探针检测特异性以及检测双链的特性。
2、在深入了解金黄色葡萄球菌生物学特性的基础上,对从医院获得的菌株,进行扩大培养并通过平板培养获得单克隆,作为后续的实验材料。
3、针对步骤1、2,结合PCR扩增技术以及磁珠提取单链技术在获得大量目标单链的基础上,实现JP探针对金黄色葡萄球菌的快速检测。
结果：1、应用JP探针检测金黄色葡萄球菌合成单链的结果表明,该方法检测金黄色葡萄球菌的可行性,建立了一种新的快速核酸检测方法。本实验通过设计三套不同的探针体系筛选出了PA1-PB1探针系统,其反应速率快,反应稳定性好,适合进行后续菌的检测；PA1-PB1探针体系优化的实验结果表明,该探针体系可以达到的检测下限是4nM,另外该探针体系可以很好的鉴别金黄色葡萄球菌以及与目标菌基因序列最相近的表皮葡萄球菌。同时合成目标单链与互补单链形成的双链探针反应,表明目前的探针体系还不能很好的检测双链,这就要求在检测临床菌时,必须制备出一定长度的单链才能有效进行。
2、通过对金黄色葡萄球菌的培养结果表明,本实验的研究菌对生长条件要求不高,在LB培养基上就可以生长良好,为后续的实验提供了大量稳定的实验材料。
3、应用JP探针检测金黄色葡萄球菌的实验表明,该方法能够快速而有效的鉴定金黄色葡萄,较传统的生化鉴定方法具有很大的优势。应用PCR扩增金黄色葡萄球菌,得到了大量的检测片段,达到第一级的放大；由于JP探针检测的目标片段是单链,为此本实验结合磁珠技术快速的提取得到了大量高纯度的单链。应用PA1-PB1探针体系检测磁珠提取单链,实现荧光信号的二级放大,从而达到仪器可检测的范围。通过以上两级放大本实验的阳性检出率达到97.25%,较单纯使用PCR技术更准确。
Purpose:1. To establish an isothermal nucleic acid detection method, Junction Probe (JP), based on restriction endonuclease, suitable for both research and clinic laboratory.
2. To develop a rapid method to detect Staphyloccocus aureus using JP technology.
Method:1. Three pairs of probes were designed and tested. By using the probes with highest reactivity and reaction reproducibility, the molar ratio of the probes was optimized, the detection specificity was investigated and the double-stranded targets were tested.
2. Based on the biological nature of Staphyloccocus aureus, the bacterial strains from hospitals were grown on LB agar and the genomic DNA were obtained by the purification of alkali treatment method.
3. Single strand DNA were obtained by combining PCR amplification and magnetic beads extraction, and Staphyloccocus aureus was detected using JP technology.
Discussion:1.The feasibility of the method to detect Staphyloccocus aureus was realized by detecting the artificial single-strand DNA target. The PA1-PB1probe pair was screened to give the fastest reaction rate and best reproducibility. The experiments showed that the detection limit of this system is4nM of temple DNA, and it can distinguish Staphylococcus aureus from Staphylococcus epidermidis, which contains SNP in the target sequence. The experiments also showed that the systems can not detect double stranded DNA, so single stranded target must be prepared while detecting clinical samples.
2.The cultivation results of Staphylococcus aureus showed that the bacteria does not need stringent growing conditions, and LB medium is good enough, which satisfied our subsequent experimental requirement.
3.This method can quickly and effectively detect Staphylococcus aureus, and has great advantages over traditional biochemical identification methods. The system has a great S/N ratio, resulting from both of the PCR and JP signal amplification in the detection of single-strand DNA samples. With the two-stage examination, the positive ratio is up to97.25%by our PCR-JP method, which is more specific than PCR technology alone.
引文
[l]王韶.应用PCR方法检测食品中金黄色葡萄球菌[D].[硕士学位论文].吉林大学,2005.
[2]王凤玲,毕丽风,侯英荣,等.金黄色葡萄球菌杀白细胞素及基因的研究进展[J].中国误诊学杂志,2007,7(21)：4693-4694.
[3]王营.金黄色葡萄球菌及其肠毒素基因分布的研究[D].[硕士学位论文].河北农业大学,2010.
[4]Atanassova V, Meindl A, Ring C. Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham-acomparison of classical culturing dete-tion and RFLP-PCR[J]. Int J Food Microbiol,2001,68(1-2):105-113.
[5]Fortunov RM, Hulten KG, Hammerman W A, et al. Evaluation and Treatment Commu-nity-Acquired Staphylococcus aureus Infections in Term and Late-Preterm PreviouHealthy Neonates[J]. Pediatrics,2007,120(5):937-945.
[6]Hawkins C, Huang J, Jin N, et al. Persistent Staphylococcus aureus Bacteremia: An Analysis of Risk Factors and Outcomes[J]. Arch Intern Med,2007,167(17):1861-1867.
[7]Hayashida A, Bartlett A H, Foster T J, et al. Staphylococcus aureus Beta-Toxin Induces Lung Injury through Syndecan-1[J]. American Journal of Pathology,2009,174:509-518.
[8]Valente A M, Jain R, Scheurer M, et al. Frequency of Infective Endocarditis Among Infants and Children With Staphylococcus aureus Bacteremia[J]. Pediatrics,2005,115(1):e15-e19.
[9]Fowler V G, Boucher H W, Corey G R, et al. Daptomycin versus Standard Therapy for Bacteremia and Endocarditis Caused by Staphylococcus aureus[J]. The new england journal of medicine,2006,355(7):653-665.
[10]杨正时,房海.人与动物病原细菌学[M].河北科学技术出版社,2003,315-331.
[11]Bean,N.H,Goulding,J.S,Lao,C and Angulo,F.J.(1996)Surveillance for foodborne-disease out-breaks United States,1988-1992. Morb.Mortal, W.Rep.45(ss-5),166.
[12]周正任主编.病原生物学[M].北京：科学出版社,2004,第二版,177
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京：科学出版社,2001,246-255.
[14]Walker GT,Fraiser MS,Schram JL.Strand displacement amplification isothermal,in vitroDNA amplifica-tion technique.Nucl Ac Res,1992,20(7):1691-1696
[15]王滨,郑向梅,高景枝,等.一起金黄色葡萄球菌毒素引起的食物中毒[J].中国卫生检验杂志,2007,17(12)：2361.
[16]吴雅儿,橙汁饮料中金黄色葡萄球菌的检测与研究,职业与健康,2000,16(11)：49
[17]Martin Dinges,Paul M.Orwin,and Patrick M.Schlievert Exotoxins of Staphylococcus aureus [J].Clinical microbiology reviews,2000,13(1):16-34
[18]Yazdankhah S P.Olsen E.Simple and direct detection of Staphylococcus aureus in milk by a tube coagulase test[J].Letters in Applied Microbiology,1998,27:111-115.
[19]陈倩,骆海朋,赵春玲等,北京市食品中五种食源性致病菌污染状况调查研究,中国卫生 检验杂志2003,10(13)：5
[20]Walker GT,Little MC,Nadeau JG.Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.Biol Sci,1992,89(1):392-396
[21]Zwadyk PJr,Down JA,Myers N,et al.Rendering of mycobacteria safe for molecular diagn-ostic studies and development of a lysis method for strand displacement amplification and PCRJ Clin microbiol,1994,32(9):2140-2146
[22]Spargo CA,Fraiser MS,Van Cleve M,et al,Detection of M.tuberculosis DNA using thermo-philic strand displacement amplification.Mol Cell probes,1996,10(4):247-256
[23]Deborah A. Wilkinson.Third Wave Technologies Invader Assays For Nucleic Acid Detection. Biol Sci,1999,13 (22):16
[24]Laura DF.The Next New Wave In Genome Analysis. Sc,1998,12(21):16
[25]Timothy J.G,Jeff H,James R.P,et al,Direct genetic analysis by matrix-assisted laser desorp-tion/ionization mass spectrometry.Proc Nztl Acad Sci USA,1999,96(11):6301-6306
[26]Timothy jG et al.Proc Natl Acad Sci USA,1999,96:6301-6306
[27]Kwiatkowski RW,Lyamichev V,Arruda M,et al.Clinical,genetic,and pharmacogenetic appl-ications of the Invader assay. Mol Diage,1999,4(4):353-364.
[28]Ashley R Connolly,Matt trau.Isothermal Detdction of DNA by Beacon-Assisted Detection Amplifiction. Communications,2010,49(15):2720-2723
[29]Lu YY,Ye SY,Hong GF.Large fragment of DNA polymerase I from Bacillus stearothermo-philus(Bst polymerase) is stable at ambient temperature. xBiotechniques,1991,11(4):464,466
[30]Mead D.A, McClary J.A, Luckey J.A,et al,Large fragment of DNA polymerase I from Baci-llus stearothermophilus (Bst polymerase) is stable at ambient temperature.Biotechniques, 1991,11(1):76-78.
[31]李翔,彭涛.一种快速等温检测核酸目标序列方法的建立.中华检验医学杂志[J],2008,3(30)：322-324
[32]Wang H,Hays JB.Preparation of DNA substrates for in vitro mismatch repair.Mol Biotech-nol,2000,15(2):92-104
[33]Higgins LS,Besnier C,Kong H.The nicking endonuclease N.BstNBI is closely related to Type Ⅱs restriction endonuclease Mlyl and Plel.Nucleic Acids Res,2001,29(12):2492-2501
[34]Gao WJ,Li X,Zeng LW,et al.Rapid isothermal detection assay:a probe amplification method for the detection of nucleic acids.Diagn Microbial Infect Dis,2008,60(2):133-141
[35]Nakayama L,Yan L,Sintim HO,et al.Junction Probes-Sequence Specific Detection of Nucleic Acid via Template Enhanced Hybridization Processes. Communication,2008,130(38), 12560-12561
[36]Sintim Herman.Probe and Methods for detection analytes[P].Patent Cooperation Treaty,Pate-nt No:PCT/US 2009/052765,2010
[37]Shizuka Nakayama, Jingxin Wang, and Herman O. Sintim.DNA-Based Peroxidation Cataly-st-What Is the Exact Role of Topology on Catalysis and Is There a Special Binding Site for Cataly-sis? Chem. Eur. J,2011,17.
[38]Shizuka Nakayama and Herman O. Sintim. Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme's Catalytic Efficiency via Probe Remodeling.2009,4.
[39]Shizuka Nakayama and Herman O. Sintim. Biomolecule detection with peroxidase-mimic-king DNAzymes; expanding detection modality with fluorogenic compounds. Molecular BioSystems,2009,9.
[40]Greenberg B. D, Newbold J. E, and Sugino A. Gene,1983 (21),33-49.
[41]Lloyd G. Mitchell and Carl R. Merril.Affinity Generation of Single-Stranded DNA for Dide-oxy Sequencing following the Polymerase Chain Reaction. Analytical Biochemistey, 1989,178:239-242
[42]Anderson S, Bankier A. T, Barrell B. G et al. Nature,1981,290:457-465.
[43]Stoflet E. S, Koeberl D. D, Sarkar G et al. Science,1988,239:491-494.
[44]Species-Level Identification of Staphylococcal Isolates by Real-Time PCR and Melt Curve Analysis. Journal of Clinical Microbiology,2005,(43)6:2876-2880
[45]Feinberg A.P. and Vogelstein.B. Anal. Biochem,1983,132:6-13.
[46]Aquadro C. F and Greenberg B. D. Genetics,1983,103:287-312.
[47]Feinberg.A.P. and Vogelstein.B. Anal. Biochem,1984,137:266-267.
[48]Chen E. J and Seeburg P. H. DNA 4,1985,165-170.
[49]Hultman.T., Stahl.S., Homes.E. and Uhlen M. Nucleic Acids Res,1989,17:4937-4946.
[50]Hattori M and Sakaki Y. Anal. B&hem,1986,152-232
[51]陈振球,章谷生.链酶亲和素的特性及其应用.上海免疫学杂志[J],1992,(12),1：56-59
[52]M.Espelund R.A,Prentice Stacy and K.S.Jakobsen.A simple method for generating single-stranded DNA probes labeled to high activities. Nucleic Acids Research,1990, (18),20:6157-6158
[2]王凤玲,毕丽风,侯英荣,等.金黄色葡萄球菌杀白细胞素及基因的研究进展[J].中国误诊学杂志,2007,7(21)：4693-4694.
[3]王营.金黄色葡萄球菌及其肠毒素基因分布的研究[D].[硕士学位论文].河北农业大学,2010.
[4]Atanassova V, Meindl A, Ring C. Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham-acomparison of classical culturing dete-tion and RFLP-PCR[J]. Int J Food Microbiol,2001,68(1-2):105-113.
[5]Fortunov RM, Hulten KG, Hammerman W A, et al. Evaluation and Treatment Commu-nity-Acquired Staphylococcus aureus Infections in Term and Late-Preterm PreviouHealthy Neonates[J]. Pediatrics,2007,120(5):937-945.
[6]Hawkins C, Huang J, Jin N, et al. Persistent Staphylococcus aureus Bacteremia: An Analysis of Risk Factors and Outcomes[J]. Arch Intern Med,2007,167(17):1861-1867.
[7]Hayashida A, Bartlett A H, Foster T J, et al. Staphylococcus aureus Beta-Toxin Induces Lung Injury through Syndecan-1[J]. American Journal of Pathology,2009,174:509-518.
[8]Valente A M, Jain R, Scheurer M, et al. Frequency of Infective Endocarditis Among Infants and Children With Staphylococcus aureus Bacteremia[J]. Pediatrics,2005,115(1):e15-e19.
[9]Fowler V G, Boucher H W, Corey G R, et al. Daptomycin versus Standard Therapy for Bacteremia and Endocarditis Caused by Staphylococcus aureus[J]. The new england journal of medicine,2006,355(7):653-665.
[10]杨正时,房海.人与动物病原细菌学[M].河北科学技术出版社,2003,315-331.
[11]Bean,N.H,Goulding,J.S,Lao,C and Angulo,F.J.(1996)Surveillance for foodborne-disease out-breaks United States,1988-1992. Morb.Mortal, W.Rep.45(ss-5),166.
[12]周正任主编.病原生物学[M].北京：科学出版社,2004,第二版,177
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京：科学出版社,2001,246-255.
[14]Walker GT,Fraiser MS,Schram JL.Strand displacement amplification isothermal,in vitroDNA amplifica-tion technique.Nucl Ac Res,1992,20(7):1691-1696
[15]王滨,郑向梅,高景枝,等.一起金黄色葡萄球菌毒素引起的食物中毒[J].中国卫生检验杂志,2007,17(12)：2361.
[16]吴雅儿,橙汁饮料中金黄色葡萄球菌的检测与研究,职业与健康,2000,16(11)：49
[17]Martin Dinges,Paul M.Orwin,and Patrick M.Schlievert Exotoxins of Staphylococcus aureus [J].Clinical microbiology reviews,2000,13(1):16-34
[18]Yazdankhah S P.Olsen E.Simple and direct detection of Staphylococcus aureus in milk by a tube coagulase test[J].Letters in Applied Microbiology,1998,27:111-115.
[19]陈倩,骆海朋,赵春玲等,北京市食品中五种食源性致病菌污染状况调查研究,中国卫生 检验杂志2003,10(13)：5
[20]Walker GT,Little MC,Nadeau JG.Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.Biol Sci,1992,89(1):392-396
[21]Zwadyk PJr,Down JA,Myers N,et al.Rendering of mycobacteria safe for molecular diagn-ostic studies and development of a lysis method for strand displacement amplification and PCRJ Clin microbiol,1994,32(9):2140-2146
[22]Spargo CA,Fraiser MS,Van Cleve M,et al,Detection of M.tuberculosis DNA using thermo-philic strand displacement amplification.Mol Cell probes,1996,10(4):247-256
[23]Deborah A. Wilkinson.Third Wave Technologies Invader Assays For Nucleic Acid Detection. Biol Sci,1999,13 (22):16
[24]Laura DF.The Next New Wave In Genome Analysis. Sc,1998,12(21):16
[25]Timothy J.G,Jeff H,James R.P,et al,Direct genetic analysis by matrix-assisted laser desorp-tion/ionization mass spectrometry.Proc Nztl Acad Sci USA,1999,96(11):6301-6306
[26]Timothy jG et al.Proc Natl Acad Sci USA,1999,96:6301-6306
[27]Kwiatkowski RW,Lyamichev V,Arruda M,et al.Clinical,genetic,and pharmacogenetic appl-ications of the Invader assay. Mol Diage,1999,4(4):353-364.
[28]Ashley R Connolly,Matt trau.Isothermal Detdction of DNA by Beacon-Assisted Detection Amplifiction. Communications,2010,49(15):2720-2723
[29]Lu YY,Ye SY,Hong GF.Large fragment of DNA polymerase I from Bacillus stearothermo-philus(Bst polymerase) is stable at ambient temperature. xBiotechniques,1991,11(4):464,466
[30]Mead D.A, McClary J.A, Luckey J.A,et al,Large fragment of DNA polymerase I from Baci-llus stearothermophilus (Bst polymerase) is stable at ambient temperature.Biotechniques, 1991,11(1):76-78.
[31]李翔,彭涛.一种快速等温检测核酸目标序列方法的建立.中华检验医学杂志[J],2008,3(30)：322-324
[32]Wang H,Hays JB.Preparation of DNA substrates for in vitro mismatch repair.Mol Biotech-nol,2000,15(2):92-104
[33]Higgins LS,Besnier C,Kong H.The nicking endonuclease N.BstNBI is closely related to Type Ⅱs restriction endonuclease Mlyl and Plel.Nucleic Acids Res,2001,29(12):2492-2501
[34]Gao WJ,Li X,Zeng LW,et al.Rapid isothermal detection assay:a probe amplification method for the detection of nucleic acids.Diagn Microbial Infect Dis,2008,60(2):133-141
[35]Nakayama L,Yan L,Sintim HO,et al.Junction Probes-Sequence Specific Detection of Nucleic Acid via Template Enhanced Hybridization Processes. Communication,2008,130(38), 12560-12561
[36]Sintim Herman.Probe and Methods for detection analytes[P].Patent Cooperation Treaty,Pate-nt No:PCT/US 2009/052765,2010
[37]Shizuka Nakayama, Jingxin Wang, and Herman O. Sintim.DNA-Based Peroxidation Cataly-st-What Is the Exact Role of Topology on Catalysis and Is There a Special Binding Site for Cataly-sis? Chem. Eur. J,2011,17.
[38]Shizuka Nakayama and Herman O. Sintim. Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme's Catalytic Efficiency via Probe Remodeling.2009,4.
[39]Shizuka Nakayama and Herman O. Sintim. Biomolecule detection with peroxidase-mimic-king DNAzymes; expanding detection modality with fluorogenic compounds. Molecular BioSystems,2009,9.
[40]Greenberg B. D, Newbold J. E, and Sugino A. Gene,1983 (21),33-49.
[41]Lloyd G. Mitchell and Carl R. Merril.Affinity Generation of Single-Stranded DNA for Dide-oxy Sequencing following the Polymerase Chain Reaction. Analytical Biochemistey, 1989,178:239-242
[42]Anderson S, Bankier A. T, Barrell B. G et al. Nature,1981,290:457-465.
[43]Stoflet E. S, Koeberl D. D, Sarkar G et al. Science,1988,239:491-494.
[44]Species-Level Identification of Staphylococcal Isolates by Real-Time PCR and Melt Curve Analysis. Journal of Clinical Microbiology,2005,(43)6:2876-2880
[45]Feinberg A.P. and Vogelstein.B. Anal. Biochem,1983,132:6-13.
[46]Aquadro C. F and Greenberg B. D. Genetics,1983,103:287-312.
[47]Feinberg.A.P. and Vogelstein.B. Anal. Biochem,1984,137:266-267.
[48]Chen E. J and Seeburg P. H. DNA 4,1985,165-170.
[49]Hultman.T., Stahl.S., Homes.E. and Uhlen M. Nucleic Acids Res,1989,17:4937-4946.
[50]Hattori M and Sakaki Y. Anal. B&hem,1986,152-232
[51]陈振球,章谷生.链酶亲和素的特性及其应用.上海免疫学杂志[J],1992,(12),1：56-59
[52]M.Espelund R.A,Prentice Stacy and K.S.Jakobsen.A simple method for generating single-stranded DNA probes labeled to high activities. Nucleic Acids Research,1990, (18),20:6157-6158
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