沙门菌条码基因研究及焦磷酸测序快速鉴定
|
摘要
在世界范围内,沙门菌是引起食物中毒最常见的致病菌,目前由沙门菌引起的食品安全、公共卫生等问题较为突出。因此建立一种准确、快速、实时、高通量的沙门菌鉴定方法对食品日常监测和沙门菌爆发疫情的调查等具有重要的意义,本研究以条码基因理论为基础并结合焦磷酸测序技术检测、鉴定沙门菌。
论文内容主要包括四部分:
1、沙门菌条码基因的初步确定
通过查阅大量中外文献,选择鉴定沙门菌最常用的invA、iroB、hns、hisJ、hilA、fimY基因作为研究对象,在Genbank数据库中下载这六个基因相应的基因序列,通过多重序列比对确定每个基因的目的片段。采用Assay design软件设计特异性PCR扩增引物和测序引物并初步确定了沙门菌的条码基因。
2、沙门菌条码基因焦磷酸测序检测方法的建立
优化PCR扩增体系和反应条件以及焦磷酸测序体系以提高本实验的可操作性。PCR扩增体系为:2×GoTaq Master Mix 12.5μl,上游引物和下游引物各5 pmol,DNA模板2μl,加无菌去离子水至25μl。反应条件为:94℃预变性5 min,94℃变性20 s,56℃退火30 s,72℃延伸20 s,35个循环,72℃延伸5 min。焦磷酸测序体系采用20μl的PCR产物加入量和优化的碱基加入程序。10株沙门菌和10株非沙门菌的测序结果显示沙门菌均测序出相应的核酸序列,非沙门菌均未测序出DNA序列,表明该方法可用于沙门菌的鉴定。对测序序列在线Blast比对分析发现测序的DNA片段均存在多态性位点,这些碱基的多样化并不会对沙门菌鉴定的准确性产生影响。
3、大量已知菌株对该方法的验证
用已建立的方法对205株沙门菌和60株非沙门菌同时进行鉴定,结果显示所有沙门菌均测序出相应的DNA序列,非沙门菌中只有一株鲍氏志贺氏菌测序结果与沙门菌hilA基因的条码基因一致,于是在沙门菌条码基因中删除了该基因以避免出现假阳性结果。通过对大量已知菌株的鉴定进一步验证了所建立检测方法的准确性和可行性,同时体现了该方法操作简便、快速、准确、高通量的特点。本实验最终确定的鉴定沙门菌的条码基因为:invA基因条码基因:TGTTAATTGCGATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCTATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCTGGTTTTATCG;TGTTGATTGCCATTAGTGCTGGTTTTATCG。iroB基因条码基因:GGAGATAACCGGCTCTCCGTCATTTTGCAG;GGAGATAACCGGCTCTCCGTCATTTGGCAG。hns基因条码基因:AGTTGTCGTTAATGAGCGTCGTGAAGAAGA;AGTTGTCGTTAACGAGCGTCGTGAAGAAGA;AGTTGTTGTTAATGAGCGTCGTGAAGAAGA。hisJ基因条码基因:CTTCTCATCTTTCACCGCCGGGCCGCCGAA;GTTCTCATCTTTCACCGCCGGGCCGGCAAA;TTTCTCATCTTTTACCGCCGGGCCGGCAAA;CTTCTCATCTTTCACCACCGGGCCGCCGAA。fimY基因条码基因:TAGCGCAAGGCGCCTCTTTAAAAGAAA;TAGCGCAAGGTGCCTCTTTAAAAGAAA;TAGCACAAGGCGCCTCTTTAAAAGAAA。
4、沙门菌条码基因焦磷酸测序检测方法的实际应用
通过在食物中毒检测和食源性疾病监测的实际应用中表明了沙门菌条码基因焦磷酸测序方法与国标法(GB/T 4789.4-2008)对沙门菌的检测结果一致,同时显示了该方法节时省力、准确、实时的优点。
Salmonella species are the major pathogenic bacteria of foodborne bacterial diseaseas in the worldwide. At present, the prominent problem is that Salmonella species is an important reason of food security and public health. Therefore, development of an accurate, rapid, real-time and high throughput identification method can significantly support thefood daily monitoring and Salmonella species outbreak investigation. This study is based on the DNA barcode theory and combines the pyrosequencing technique in order to identify Salmonella species.
Four parts were included in this thesis:
Firstly, Salmonella DNA barcode was preliminarily determined. invA, iroB, hns, hisJ, hilA, fimY gene which the most commonly used to identify Salmonella were selected from the sino-foreign literature. The six genes corresponding sequences were download in Genbank database and multiple sequence alignment determine the purpose fragments of each gene. The six specific PCR primer sets and sequencing primers were designed using Assay design software and preliminarily determined the DNA barcode.
Secondly, Salmonella DNA barcode pyrosequencing method was established and Salmonella DNA barcode sequence was determined.
Through optimizing the PCR amplification system and PCR reaction conditions and pyrosequencing conditions, the experiment maneuverability were enhanced. PCR mixture contained the following: 12.5μl 2×Go Taq Master Mix, 5pmol of each primer, 2μl of DNA template and added ddH2O until 25μl. The PCR reaction: after initial denaturation at 94°C for 5 min, 35 cycles of amplification included denaturation at 94°C for 20 s, annealing at 56°C for 30 s, and elongation at 72°C for 20 s. The reaction concluded with a 5 min extension phase at 72°C. 20μl of biotinylated PCR products and optimition of bases join procedure were used in the pyrosequencing system. 10 Salmonella strains and 10 non-Salmonella strains by prosequencing showed that only all of Salmonella strains obtained the correct DNA sequences. Some polymorphism sites were founded in the DNA sequence by online Blast tool analysis, but the base variation will not affect the accuracy of Salmonella detection.
Thirdly, the new mthod were verified by a large number of known strains.
205 Salmonella strains and 60 non-Salmonella strains were identified by this newmethod, and the result showed that all Salmonella strains obtained DNA sequence which correspond with Salmonella DNA barcode. In non-Salmonella strains, only Shigella boydii DNA sequence was compatibile with the hilA DNA barcode of Salmonella. To avoid the false positive result, hilA gene was excluded in Salmonella identification barcode gene panel. Through a large number of strains further verified the accuracy and feasibility of this method. It indicated that pyrosequencing was a easy to operate, rapid, accurate, sensitive and high-throughput method. So the DNA barcode sequence to identify the Salmonella was determined in the study: (A)invA gene barcode:TGTTAATTGCGATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCTATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCTGGTTTTATCG;TGTTGATTGCCATTAGTGCTGGTTTTATCG.(B)iroB gene barcode:GGAGATAACCGGCTCTCCGTCATTTTGCAG;GGAGATAACCGGCTCTCCGTCATTTGGCAG.(C)hns gene barcode:AGTTGTCGTTAATGAGCGTCGTGAAGAAGA;AGTTGTCGTTAACGAGCGTCGTGAAGAAGA;AGTTGTTGTTAATGAGCGTCGTGAAGAAGA。(D)hisJ gene barcode:CTTCTCATCTTTCACCGCCGGGCCGCCGAA;GTTCTCATCTTTCACCGCCGGGCCGGCAAA;TTTCTCATCTTTTACCGCCGGGCCGGCAAA;CTTCTCATCTTTCACCACCGGGCCGCCGAA。(E)fimY gene barcode:TAGCGCAAGGCGCCTCTTTAAAAGAAA;TAGCGCAAGGTGCCTCTTTAAAAGAAA;TAGCACAAGGCGCCTCTTTAAAAGAAA。
Finally, practical application of Salmonella DNA barcode pyrosequencing method.
Through practical application which the food poisoning detection and food-borne disease monitoring, it showed that the result by Salmonella DNA barcode pyrosequencing method was same to the traditional cultural method (GB/T GB 4789.4-2008), and showed advantages of this method: efficiency, accuracy, real time.
论文内容主要包括四部分:
1、沙门菌条码基因的初步确定
通过查阅大量中外文献,选择鉴定沙门菌最常用的invA、iroB、hns、hisJ、hilA、fimY基因作为研究对象,在Genbank数据库中下载这六个基因相应的基因序列,通过多重序列比对确定每个基因的目的片段。采用Assay design软件设计特异性PCR扩增引物和测序引物并初步确定了沙门菌的条码基因。
2、沙门菌条码基因焦磷酸测序检测方法的建立
优化PCR扩增体系和反应条件以及焦磷酸测序体系以提高本实验的可操作性。PCR扩增体系为:2×GoTaq Master Mix 12.5μl,上游引物和下游引物各5 pmol,DNA模板2μl,加无菌去离子水至25μl。反应条件为:94℃预变性5 min,94℃变性20 s,56℃退火30 s,72℃延伸20 s,35个循环,72℃延伸5 min。焦磷酸测序体系采用20μl的PCR产物加入量和优化的碱基加入程序。10株沙门菌和10株非沙门菌的测序结果显示沙门菌均测序出相应的核酸序列,非沙门菌均未测序出DNA序列,表明该方法可用于沙门菌的鉴定。对测序序列在线Blast比对分析发现测序的DNA片段均存在多态性位点,这些碱基的多样化并不会对沙门菌鉴定的准确性产生影响。
3、大量已知菌株对该方法的验证
用已建立的方法对205株沙门菌和60株非沙门菌同时进行鉴定,结果显示所有沙门菌均测序出相应的DNA序列,非沙门菌中只有一株鲍氏志贺氏菌测序结果与沙门菌hilA基因的条码基因一致,于是在沙门菌条码基因中删除了该基因以避免出现假阳性结果。通过对大量已知菌株的鉴定进一步验证了所建立检测方法的准确性和可行性,同时体现了该方法操作简便、快速、准确、高通量的特点。本实验最终确定的鉴定沙门菌的条码基因为:invA基因条码基因:TGTTAATTGCGATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCTATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCTGGTTTTATCG;TGTTGATTGCCATTAGTGCTGGTTTTATCG。iroB基因条码基因:GGAGATAACCGGCTCTCCGTCATTTTGCAG;GGAGATAACCGGCTCTCCGTCATTTGGCAG。hns基因条码基因:AGTTGTCGTTAATGAGCGTCGTGAAGAAGA;AGTTGTCGTTAACGAGCGTCGTGAAGAAGA;AGTTGTTGTTAATGAGCGTCGTGAAGAAGA。hisJ基因条码基因:CTTCTCATCTTTCACCGCCGGGCCGCCGAA;GTTCTCATCTTTCACCGCCGGGCCGGCAAA;TTTCTCATCTTTTACCGCCGGGCCGGCAAA;CTTCTCATCTTTCACCACCGGGCCGCCGAA。fimY基因条码基因:TAGCGCAAGGCGCCTCTTTAAAAGAAA;TAGCGCAAGGTGCCTCTTTAAAAGAAA;TAGCACAAGGCGCCTCTTTAAAAGAAA。
4、沙门菌条码基因焦磷酸测序检测方法的实际应用
通过在食物中毒检测和食源性疾病监测的实际应用中表明了沙门菌条码基因焦磷酸测序方法与国标法(GB/T 4789.4-2008)对沙门菌的检测结果一致,同时显示了该方法节时省力、准确、实时的优点。
Salmonella species are the major pathogenic bacteria of foodborne bacterial diseaseas in the worldwide. At present, the prominent problem is that Salmonella species is an important reason of food security and public health. Therefore, development of an accurate, rapid, real-time and high throughput identification method can significantly support thefood daily monitoring and Salmonella species outbreak investigation. This study is based on the DNA barcode theory and combines the pyrosequencing technique in order to identify Salmonella species.
Four parts were included in this thesis:
Firstly, Salmonella DNA barcode was preliminarily determined. invA, iroB, hns, hisJ, hilA, fimY gene which the most commonly used to identify Salmonella were selected from the sino-foreign literature. The six genes corresponding sequences were download in Genbank database and multiple sequence alignment determine the purpose fragments of each gene. The six specific PCR primer sets and sequencing primers were designed using Assay design software and preliminarily determined the DNA barcode.
Secondly, Salmonella DNA barcode pyrosequencing method was established and Salmonella DNA barcode sequence was determined.
Through optimizing the PCR amplification system and PCR reaction conditions and pyrosequencing conditions, the experiment maneuverability were enhanced. PCR mixture contained the following: 12.5μl 2×Go Taq Master Mix, 5pmol of each primer, 2μl of DNA template and added ddH2O until 25μl. The PCR reaction: after initial denaturation at 94°C for 5 min, 35 cycles of amplification included denaturation at 94°C for 20 s, annealing at 56°C for 30 s, and elongation at 72°C for 20 s. The reaction concluded with a 5 min extension phase at 72°C. 20μl of biotinylated PCR products and optimition of bases join procedure were used in the pyrosequencing system. 10 Salmonella strains and 10 non-Salmonella strains by prosequencing showed that only all of Salmonella strains obtained the correct DNA sequences. Some polymorphism sites were founded in the DNA sequence by online Blast tool analysis, but the base variation will not affect the accuracy of Salmonella detection.
Thirdly, the new mthod were verified by a large number of known strains.
205 Salmonella strains and 60 non-Salmonella strains were identified by this newmethod, and the result showed that all Salmonella strains obtained DNA sequence which correspond with Salmonella DNA barcode. In non-Salmonella strains, only Shigella boydii DNA sequence was compatibile with the hilA DNA barcode of Salmonella. To avoid the false positive result, hilA gene was excluded in Salmonella identification barcode gene panel. Through a large number of strains further verified the accuracy and feasibility of this method. It indicated that pyrosequencing was a easy to operate, rapid, accurate, sensitive and high-throughput method. So the DNA barcode sequence to identify the Salmonella was determined in the study: (A)invA gene barcode:TGTTAATTGCGATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCGATTAGTGCCGGTTTTATCG;TGTTGATTGCTATTAGTGCCGGTTTTATCG;TGCTGATTGCGATTAGTGCTGGTTTTATCG;TGTTGATTGCCATTAGTGCTGGTTTTATCG.(B)iroB gene barcode:GGAGATAACCGGCTCTCCGTCATTTTGCAG;GGAGATAACCGGCTCTCCGTCATTTGGCAG.(C)hns gene barcode:AGTTGTCGTTAATGAGCGTCGTGAAGAAGA;AGTTGTCGTTAACGAGCGTCGTGAAGAAGA;AGTTGTTGTTAATGAGCGTCGTGAAGAAGA。(D)hisJ gene barcode:CTTCTCATCTTTCACCGCCGGGCCGCCGAA;GTTCTCATCTTTCACCGCCGGGCCGGCAAA;TTTCTCATCTTTTACCGCCGGGCCGGCAAA;CTTCTCATCTTTCACCACCGGGCCGCCGAA。(E)fimY gene barcode:TAGCGCAAGGCGCCTCTTTAAAAGAAA;TAGCGCAAGGTGCCTCTTTAAAAGAAA;TAGCACAAGGCGCCTCTTTAAAAGAAA。
Finally, practical application of Salmonella DNA barcode pyrosequencing method.
Through practical application which the food poisoning detection and food-borne disease monitoring, it showed that the result by Salmonella DNA barcode pyrosequencing method was same to the traditional cultural method (GB/T GB 4789.4-2008), and showed advantages of this method: efficiency, accuracy, real time.
引文
[1] Centers for Disease Control and Prevention (CDC). Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food-10 states, 2009[J]. MMWR Morb Mortal Wkly Rep, 2010, 59(14): 418-422.
[2]余贺.医学微生物学[M].北京:人民卫生出版社,1983,303-305.
[3]俞树荣.微生物学和微生物学检验:第2版[M].北京;人民卫生出版社,1988,175-177.
[4] Manzano M, Cocolin L, Astori G, Pipan C, Botta GA, Cantoni C, Comi G. Development of a PCR microplate-capture hybridization method for simple,fast and sensitive detection of Salmonella serovars in food[J]. Molecular and Cellular Probes, 1998, 12(4): 227-234.
[5] Whyte P, Mc Gill K, Collins JD, Gormley E. The prevalence and PCR detection of Salmonella contamination in raw poultry[J]. Veterinary Microbiology, 2002, 89(1): 53-60.
[6] Keusch, GT. Systemic gastro-intestinal infections: a clinical overview. In Molecular Medical Microbiology2 [M]. Edited by M. Sussman. San Diego: Academic Press, 2002, 1357-1363.
[7] Humphrey T. Public-health aspects of Salmonella infection. Salmonella in Domestic Animals (Way C & Way A,eds)[M]. CABI Publishing, Oxon, UK. 2000, 245-263.
[8] Rijpens N, Herman L, Vereecken F, Jannes G, De Smedt J, De Zutter L. Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR[J]. Int J Food Microbiol, 1999, 46(1): 37-44.
[9] Whyte P, Mc Gill K, Collins JD, Gormley E. The prevalence and PCR detection of Salmonella contamination in raw poultry[J]. Vet Microbiol, 2002, 89(1): 53-60.
[10] Stone GG, Oberst RD, Hays MP, McVey S, Chengappa MM. Detection of Salmonella serovars fromclinical samples by enrichment broth cultivation-PCR procedure[J]. J Clin Microbiol, 1994, 32(7):1742-1749.
[11] Carter G R, Chengappa M M. Enterobacteriaceae, In Essentials of veterinary bacteriology and mycology[M]. Lea and Febiger, Philadelphia. 1991, 150-164.
[12] Van der Zee, H. & Huis in’t Veld, J. H. J. Methods for the rapid detection of Salmonella. In Salmonella in Domestic Animals[M]. Edited by C. Wray & A. Wray. Wallingford, UK: CABI Publishing, 2000, 373-391.
[13] AOAC/BAM(Bacteriological Analytical Manual,FDA).伯杰细菌鉴定手册(第八版)[M].科学出版社,1984.
[14] GB/T 4789.4-2008.食品卫生微生物学检验沙门菌检验[S].中国标准出版社,2008.
[15] Odumeru JA, Steele M, Fruhner L, Larkin C, Jiang J, Mann E, McNab WB. Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems[J]. A J Clin Microbiol, 1999, 37(4): 944-999.
[16]杜春明,郭云昌,刘秀梅. API 20E、PCR方法在沙门氏菌鉴定中的应用[J].实用预防医学,2005,12(5):1006-1007.
[17]杨毓环,陈伟伟. VITEK全自动微生物检测系统原理及其应用[J].海峡预防医学杂志,2000,6(3):38-39.
[18] Yoshimasu MA, Zawistowski J. Application of rapid dot blot immunoassay for detection of Salmonella enterica serovar enteritidis in eggs, poultry, and other foods[J]. Appl EnvironMicrobiol, 2001, 67(1) :459.
[19] Janyapoon K, Korbsrisate S, Thamapa H, Thongmin S, Kanjanahareutai S, Wongpredee N, Sarasombath S. Rapid detection of Salmonella enterica serovar Choleraesuis in blood cultures by a dot blot enzyme-linked immunosorbent assay[J]. Clin Diagn Lab Immunol, 2000, 7(6): 977-979.
[20] Cloak OM, Duffy G, Sheridan JJ, McDowell DA, Blair IS. Development of a surface adhesion immunofluorescent technique for the rapid detection of Salmonella spp. from meat and poultry[J]. J Appl Microbiol, 1999, 86(4): 583-590.
[21]文其乙,焦新安,刘秀梵,张如宽,杨静.直接ELISA检测沙门氏菌方法的建立及其应用研究[J].中国兽医学报,1995,15(2):105-111.
[22]曹春梅,焦新安,刘海侠.斑点免疫金渗滤法快速检测沙门氏菌09抗原的研究[J].中国人兽共患病杂志,2005,21(3):210-212.
[23] Che YH, Li Y, Slavik M, Paul D. Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method[J]. J Food Prot, 2000, 63(8): 1043-1048.
[24] Brooks JL, Mirhabibollahi B, Kroll RG. Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods[J]. J Appl Bacteriol, 1992, 73(3):189-196.
[25]曹同雪,唐中令. SPA协凝试验法快速检验肉中沙门氏菌[J].西南民族学院学报(自然科学版),1997,23(3):284-288.
[26]应成玉,杨生栋,申什菊,曹迎春,刘武.乳胶凝集试验检测成品饲料中的沙门氏菌[J].青海畜牧兽医杂志,2006,36(2):14-15.
[27]林涛,兰敏,黄伟,冯小军,王静,杨雪娇.荧光酶免疫分析仪-微生物鉴定仪在水产品沙门氏菌检测中的应用[J].分析与检验,2008,34(8):133-136.
[28] Curiale MS, Gangar V, Gravens C. VIDAS enzyme-linked fluorescent immunoassay for detection of Salmonella in foods: collaborative study[J]. J AOAC Int, 1997, 80(3): 491-504.
[29] Fitts R, Diamond M, Hamilton C, Neri M. DNA-DNA hybridization assay for detection of Salmonella spp. in foods[J]. Appl Environ Microbiol, 1983, 46(5): 1146-1151.
[30] Cohen ND, McGruder ED, Neibergs HL, Behle RW, Wallis DE, Hargis BM. Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella[J]. Poult Sci, 1994, 73(2): 354-357.
[31] O'Regan E, McCabe E, Burgess C, McGuinness S, Barry T, Duffy G, Whyte P, Fanning S. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples[J]. BMC Microbiol, 2008, 8:156.
[32] Malorny B, Bunge C, Helmuth R. A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs[J]. J Microbiol Methods, 2007, 70(2): 245-251.
[33] Chen J, Zhang L, Paoli GC, Shi C, Tu SI, Shi X. A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis[J]. Int J Food Microbiol, 2010, 137(2-3): 168-174.
[34] KrascsenicsováK, PiknováL, KaclíkováE, Kuchta T. Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction[J]. Lett Appl Microbiol, 2008, 46(4): 483-487.
[35] Trafny EA, Koz?owska K, Szpakowska M. A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces[J]. Lett Appl Microbiol, 2006, 43(6): 673-679.
[36] Saroj SD, Shashidhar R, Karani M, Bandekar JR. Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture - nested PCR combination assay[J]. Mol Cell Probes, 2008, 22(3): 201-206.
[37] Bansal NS, Gray V, McDonell F. Validated PCR assay for the routine detection of Salmonella in food[J]. J Food Prot, 2006, 69(2): 282-287.
[38] Kawasaki S, Fratamico PM, Horikoshi N, Okada Y, Takeshita K, Sameshima T, Kawamoto S. Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing[J]. Foodborne Pathog Dis, 2009, 6(1): 81-89.
[39] Woo PC, Leung PK, Leung KW, Yuen KY. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient[J]. Mol Pathol, 2000, 53(4): 211-215.
[40] Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P. Real-time DNA sequencing using detection of pyrophosphate release[J]. Anal Biochem, 1996, 242(1): 84-89.
[41] Agah A, Aghajan M, Mashayekhi F, Amini S, Davis RW, Plummer JD, Ronaghi M, Griffin PB. A multi-enzyme model for Pyrosequencing[J]. Nucleic acids research, 2004, 32(21): 166.
[42] Elahi E, Ronaghi M. Pyrosequencing: a tool for DNA sequencing analysis[J].Methods in molecular biology, 2004, 255(1): 211-219.
[43] Nyrén P, Lundin A. Enzymatic method for continuos monitoring of inorganic pyrophosphate synthesis[J]. Analyt Biochem, 1985, 151(2): 504-509.
[44] Nyrén P. Enzymatic method for continuous monitoring of DNA polymerase activity[J]. Anal Biochem, 1987, 167(2): 235-238.
[45] Nyrén P, Karamohamed S, Ronaghi M. Detection of single-base changes using a bioluminometric primer extension assay[J]. Anal Biochem, 1997, 244(2): 367-373.
[46] Hyman E D. A new method of sequencing DNA[J]. Analyt Biochem, 1988, 174(2): 423-436.
[47] Ronaghi M, Uhlén M, Nyrén P. A sequencing method based on real-time pyrophosphate[J]. Science, 1998, 281(5375): 363-365.
[48] Ronaghi M, Elahi E. Discovery of single nucleotid polymorphisms and mutations by pyrosequencing[J]. Comp Funct Genomics, 2002, 3(1): 51-56.
[49] Langaee T, Ronaghi M. Genetic variation analyses by Pyrosequencing[J]. Mutat Res, 2005, 573(1-2): 96-102.
[50] Klenow H, Overgaard-Hansen K, Patkar SA. Proteolytic cleavage fonative DNA polymerase into two different catalytic fragments.Influence of assay condtions on the change of exonuclease activity and polymerase activity accompanying cleavage[J]. Eur J Biochem, 1971, 22(3): 371-381.
[51] Segel IH, Renosto F, Seubert PA. Sulfate-activating enzymes[J]. Methods Enzymol, 1987, 143:334-349.
[52] Deluca M. Firefly luciferase[J]. Adv Enzymol Relat Areas Mol Biol, 1976, 44:37-68.
[53] Komoszynski M, Wojtczak A. Apyrases (ATP diphosphohydrolases, EC 3.6.1.5): function and relationship to ATPases[J]. Biochim Biophys Acta, 1996, 1310: 233-241.
[54] Ahmadian A, Ehn M, Hober S. Pyrosequencing: History, biochemistry and future[J]. Clin Chim Acta, 2006, 363(1-2): 83-94.
[55] James D Watson. The Human Genome Project: Past, Present, and Future[J]. Science, 1990, 248(4951): 44-49.
[56] David R Bentley. The Human Genome Project-An Overview[J]. Medicinal Research Reviews, 2000, 20(3): 189-196.
[57] Gharizadeh B, Herman ZS, Eason RG, Jejelowo O, Pourmand N. Large-scale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing[J]. Electrophoresis, 2006, 27(15): 3042-3047.
[58] Ronaghi M. Pyrosequencing sheds light on DNA sequencing[J]. Genome Res, 2001, 11(1): 3-11.
[59] Gharizadeh B, Nordstr?m T, Ahmadian A, Ronaghi M, Nyrén P. Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate)Sp-isomer[J]. Anal Biochem, 2002, 301(1): 82-90.
[60] Chen DC, Saarela J, Nuotio I, Jokiaho A, Peltonen L, Palotie A. Comparison of GenFlex Tag array and pyrosequencing in SNP genotyping[J]. J. Mol. Diagn, 2003, 5(4): 243-249.
[61] Lavebratt C, Sengul S. Single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools using Pyrosequencing[J]. Nat Protoc, 2006, 1(6): 2573-2582.
[62] Royo JL, Galán JJ. Pyrosequencing for SNP genotyping[J]. Methods Mol Biol, 2009, 578(3): 123-133.
[63] Lin YS, Liu FG, Wang TY, Pan CT, Chang WT, Li WH. A simple method using PyrosequencingTM to identify de novo SNPs in pooled DNA samples[J]. Nucleic Acids Res, 2010, 3:1-11.
[64] Narasimhan S, Falkenberg VR, Khin MM, Rajeevan MS. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing[J]. BMC Res Notes, 2009, 12(2): 104-112.
[65] Tost J, Gut IG. DNA methylation analysis by pyrosequencing[J]. Nat Protoc, 2007, 2(9): 2265-2275.
[66] Nordstr?m T, Gharizadeh B, Pourmand N, Nyren P, Ronaghi M. Method enabling fast partial sequencing of cDNA clones[J]. Anal Biochem, 2001, 292(2): 266-271.
[67] Garcia CA, Ahmadian A, Gharizadeh B, Lundeberg J, Ronaghi M, Nyrén P. Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene[J]. Gene, 2000, 253(2): 249-257.
[68] Sinclair A, Arnold C, Woodford N. Rapid detection and estimation by pyrosequencing of 23S rRNA genes with a single nucleotide polymorphism conferring linezolid resistance in Enterococci[J]. Antimicrob Agents Chemother[J]. 2003, 47(11): 3620-3622.
[69] Divne AM, Edlund H, Allen M. Forensic analysis of autosomal STR markers using Pyrosequencing [J]. Forensic Sci Int Genet, 2010, 4(2): 122-129.
[70]袁建林,武国军,薛丽,赵锦荣,白玉杰,杨芳,王禾,张运涛,杨力军.应用焦磷酸微测序技术行HLA-DRB基因型分析[J].中国现代医学杂志,2006,16(8):1164-1170.
[71]杨会勇,那广水,朱术会,邹秉杰,奚涛,周国华.利用线性指数PCR-焦磷酸测序技术快速测3种海洋弧菌[J].中国药科大学学报,2009,40(5):460-46.
[72]孟成艳,金嘉琳,雷建强,王莹,张舒,张文宏. PCR-焦磷酸测序法快速检测和鉴定临床常见致病菌的研究[J].中国医学检验杂志,2007,30(4):456-458.
[73]毛静月.焦磷酸测序技术在高危型HpV分型中应用的研[D].第二军医大学硕士学位论文,2008,6.
[74]姚蓝.乙型肝炎病毒Pre-C/BCP突变热点的焦磷酸测序方法的建立及其可靠性研究[D].中山大学硕士学位论文,2007,5.
[75]李秀娟,徐保红,宋红梅,李丽婕,田会芳.沙门菌PCR-焦磷酸测序法检测[J].中国公共卫生,2009,25(8):997-998.
[76]田会方,徐保红,李秀娟,李波,王晓丽. PCR-焦磷酸测序技术在沙门菌食物中毒中的应用[J].中国卫生检验杂志,2009,19(11):2584-2585.
[77] Pai R, Limor J, Beall B. Use of pyrosequencing to differentiate Streptococcus pneumoniae serotypes 6A and 6B[J]. J Clin Microbiol, 2005, 43(9): 4820-4822.
[78] Unnerstad H, Ericsson H, Alderborn A, Tham W, Danielsson-Tham ML, Mattsson JG. Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene[J]. Appl Environ Microbiol, 2001, 67(11): 5339-5342.
[79] Gharizadeh B, Norberg E, L?ffler J, Jalal S, Tollemar J, Einsele H, Klingspor L, Nyrén P. Identification of medically important fungi by the Pyrosequencing technology[J]. Mycoses, 2004 ,47(1-2): 29-33.
[80] Monstein H, Nikpour-Badr S, Jonasson J. Rapid molecular identification and subtyping of Helicobacter pylori by pyrosequencing of the 16S rDNA variable V1 and V3 regions[J]. FEMS Microbiol Lett, 2001, 199(1): 103-107.
[81] Gharizadeh B, Kalantari M, Garcia CA, Johansson B, Nyrén P. Typing of human papillomavirus by pyrosequencing[J]. Lab Invest, 2001, 81(5): 673-679.
[82] Elahi E, Pourmand N, Chaung R, Rofoogaran A, Boisver J, Samimi-Rad K, Davis RW, Ronaghi M. Determination of hepatitis C virus genotype by Pyrosequencing[J]. J Virol Methods, 2003, 109(2): 171-176.
[83] Wahab T, Hjalmarsson S, Wollin R, Engstrand L. Pyrosequencing Bacillus anthracis[J]. Emerg Infect Dis, 2005, 11(10): 1527-1531.
[84] Kobayashi H, Hall GS, Tuohy MJ, Knothe U, Procop GW, Bauer TW. Bilateral periprosthetic joint infection caused by Salmonella enterica serotype Enteritidis, and identification of Salmonella sp using molecular techniques[J]. Int J Infect Dis, 2009, 13(6): e463-e466.
[85] Hebert PD, Ratnasingham S, deWaard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[J]. Proc Biol Sci, 2003, 270(1): 96-99.
[86] Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH. Use of DNA barcodes to identify flowering plants[J]. Proc Natl Acad Sci USA, 2005, 102(23): 8369-8374.
[87] Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding[J]. Nucleic Acids Res, 2007, 35(3): e14.
[88] Schindel DE, Miller SE. DNA barcoding a useful tool for taxonomists[J]. Nature, 2005, 435(7038): 17.
[89] Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes[J]. Proc Biol Sci, 2003, 270(1512): 313-321.
[90] Waugh J. DNA barcoding in animal species: progress, potential and pitfalls[J]. Bioessays, 2007, 29(2): 188-197.
[91] Aliabadian M, Kaboli M, Nijman V, Vences M. Molecular identification of birds: performance of distance-based DNA barcoding in three genes to delimit parapatric species[J]. PLoS One, 2009, 4(1): e4119.
[92] CBOL Plant Working Group. A DNA barcode for land plants[J]. Proc Natl Acad Sci USA, 2009, 106(31): 12794-12797.
[93] Kim HJ, Park SH, Lee TH, Nahm BH, Chung YH, Seo KH, Kim HY. Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars[J]. J Food Prot, 2006, 69(7): 1653-1661.
[94] Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galán JE, Ginocchio C, Curtiss R 3rd, Gyles CL. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J]. Molecular and Cellular Probes, 1992, 6(4): 271-279.
[95] Bülte M, Jakob P. The use of a PCR-generated invA probe for the detection of Salmonella spp. in artificially and naturally contaminated foods[J]. Int J Food Microbiol, 1995, 26(3): 335-344.
[96] JeníkováG, PazlarováJ, DemnerováK. Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay[J]. Int Microbiol, 2000, 3(4): 225-229.
[97] Chiu CH, Ou JT. Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay[J]. J Clin Microbiol, 1996, 34(10): 2619-2622.
[98] Cocolin L, Manzano M, Cantoni C, Comi G. Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food[J]. J Appl Microbiol, 1998, 85(4): 673-677.
[99] B?umler AJ, Tsolis RM, van der Velden AW, Stojiljkovic I, Anic S, Heffron F. Identification of a new iron regulated locus of Salmonella typhi[J]. Gene, 1996, 183(1-2): 207-213.
[100] B?umler AJ, Heffron F, Reissbrodt R. Rapid detection of Salmonella enterica with primers specific for iroB[J]. J Clin Microbiol, 1997, 35(5): 1224-1230.
[101] Jones DD, Law R, Bej AK. Detection of Salmonella spp. in oysters using polymerase chain reaction (PCR) and gene probes[J]. J. Food Sci, 1993, 58: 1191-1197.
[102] Higgins CF, Haag PD, Nikaido K, Ardeshir F, Garcia G, Ames GF. Complete nucleotide sequence and identification of membrane components of the histidine transport operon of S. typhimurium[J]. Nature, 1982, 298(5876): 723-727.
[103] Mills DM, Bajaj V, Lee CA. A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome[J]. Mol Microbiol, 1995, 15(4): 749-759.
[104] Galán JE. Molecular genetic bases of Salmonella entry into host cells[J]. Mol Microbiol, 1996, 20(2): 263-271.
[105] Bajaj V, Hwang C, Lee CA. hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes[J]. Mol Microbiol, 1995, 18(4): 715-727.
[106] Duguid JP, Smith IW, Dempster G, Edmunds PN. Non-flagellar filamentous appendages (fimbriae) and haemagglutinating activity in Bacterium coli[J]. J Pathol Bacteriol, 1955, 70(2): 335-348.
[107] Clegg S, Swenson DL. Fimbriae: Adhesion, Genetics, Biogenesis, and Vaccines. CRC Press, BocaRaton, FL, In: Klemm, P. (Ed.), 1994, 105-113.
[108] Yeh KS, Chen TH, Liao CW, Chang CS, Lo HC. PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species[J]. Int J Food Microbiol, 2002, 78(3):227-234.
[109] Anonymous. Microbiology of Food and Animal Feeding Stuffs Protocol for the Validation of Alternative Methods (ISO16140:2003). European Committee for Standardization, 2003.
[110] Malorny B, Hoorfar J, Bunge C, Helmuth R. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard[J]. Appl Environ Microbiol, 2003, 69(1): 290-296.
[2]余贺.医学微生物学[M].北京:人民卫生出版社,1983,303-305.
[3]俞树荣.微生物学和微生物学检验:第2版[M].北京;人民卫生出版社,1988,175-177.
[4] Manzano M, Cocolin L, Astori G, Pipan C, Botta GA, Cantoni C, Comi G. Development of a PCR microplate-capture hybridization method for simple,fast and sensitive detection of Salmonella serovars in food[J]. Molecular and Cellular Probes, 1998, 12(4): 227-234.
[5] Whyte P, Mc Gill K, Collins JD, Gormley E. The prevalence and PCR detection of Salmonella contamination in raw poultry[J]. Veterinary Microbiology, 2002, 89(1): 53-60.
[6] Keusch, GT. Systemic gastro-intestinal infections: a clinical overview. In Molecular Medical Microbiology2 [M]. Edited by M. Sussman. San Diego: Academic Press, 2002, 1357-1363.
[7] Humphrey T. Public-health aspects of Salmonella infection. Salmonella in Domestic Animals (Way C & Way A,eds)[M]. CABI Publishing, Oxon, UK. 2000, 245-263.
[8] Rijpens N, Herman L, Vereecken F, Jannes G, De Smedt J, De Zutter L. Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR[J]. Int J Food Microbiol, 1999, 46(1): 37-44.
[9] Whyte P, Mc Gill K, Collins JD, Gormley E. The prevalence and PCR detection of Salmonella contamination in raw poultry[J]. Vet Microbiol, 2002, 89(1): 53-60.
[10] Stone GG, Oberst RD, Hays MP, McVey S, Chengappa MM. Detection of Salmonella serovars fromclinical samples by enrichment broth cultivation-PCR procedure[J]. J Clin Microbiol, 1994, 32(7):1742-1749.
[11] Carter G R, Chengappa M M. Enterobacteriaceae, In Essentials of veterinary bacteriology and mycology[M]. Lea and Febiger, Philadelphia. 1991, 150-164.
[12] Van der Zee, H. & Huis in’t Veld, J. H. J. Methods for the rapid detection of Salmonella. In Salmonella in Domestic Animals[M]. Edited by C. Wray & A. Wray. Wallingford, UK: CABI Publishing, 2000, 373-391.
[13] AOAC/BAM(Bacteriological Analytical Manual,FDA).伯杰细菌鉴定手册(第八版)[M].科学出版社,1984.
[14] GB/T 4789.4-2008.食品卫生微生物学检验沙门菌检验[S].中国标准出版社,2008.
[15] Odumeru JA, Steele M, Fruhner L, Larkin C, Jiang J, Mann E, McNab WB. Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems[J]. A J Clin Microbiol, 1999, 37(4): 944-999.
[16]杜春明,郭云昌,刘秀梅. API 20E、PCR方法在沙门氏菌鉴定中的应用[J].实用预防医学,2005,12(5):1006-1007.
[17]杨毓环,陈伟伟. VITEK全自动微生物检测系统原理及其应用[J].海峡预防医学杂志,2000,6(3):38-39.
[18] Yoshimasu MA, Zawistowski J. Application of rapid dot blot immunoassay for detection of Salmonella enterica serovar enteritidis in eggs, poultry, and other foods[J]. Appl EnvironMicrobiol, 2001, 67(1) :459.
[19] Janyapoon K, Korbsrisate S, Thamapa H, Thongmin S, Kanjanahareutai S, Wongpredee N, Sarasombath S. Rapid detection of Salmonella enterica serovar Choleraesuis in blood cultures by a dot blot enzyme-linked immunosorbent assay[J]. Clin Diagn Lab Immunol, 2000, 7(6): 977-979.
[20] Cloak OM, Duffy G, Sheridan JJ, McDowell DA, Blair IS. Development of a surface adhesion immunofluorescent technique for the rapid detection of Salmonella spp. from meat and poultry[J]. J Appl Microbiol, 1999, 86(4): 583-590.
[21]文其乙,焦新安,刘秀梵,张如宽,杨静.直接ELISA检测沙门氏菌方法的建立及其应用研究[J].中国兽医学报,1995,15(2):105-111.
[22]曹春梅,焦新安,刘海侠.斑点免疫金渗滤法快速检测沙门氏菌09抗原的研究[J].中国人兽共患病杂志,2005,21(3):210-212.
[23] Che YH, Li Y, Slavik M, Paul D. Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method[J]. J Food Prot, 2000, 63(8): 1043-1048.
[24] Brooks JL, Mirhabibollahi B, Kroll RG. Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods[J]. J Appl Bacteriol, 1992, 73(3):189-196.
[25]曹同雪,唐中令. SPA协凝试验法快速检验肉中沙门氏菌[J].西南民族学院学报(自然科学版),1997,23(3):284-288.
[26]应成玉,杨生栋,申什菊,曹迎春,刘武.乳胶凝集试验检测成品饲料中的沙门氏菌[J].青海畜牧兽医杂志,2006,36(2):14-15.
[27]林涛,兰敏,黄伟,冯小军,王静,杨雪娇.荧光酶免疫分析仪-微生物鉴定仪在水产品沙门氏菌检测中的应用[J].分析与检验,2008,34(8):133-136.
[28] Curiale MS, Gangar V, Gravens C. VIDAS enzyme-linked fluorescent immunoassay for detection of Salmonella in foods: collaborative study[J]. J AOAC Int, 1997, 80(3): 491-504.
[29] Fitts R, Diamond M, Hamilton C, Neri M. DNA-DNA hybridization assay for detection of Salmonella spp. in foods[J]. Appl Environ Microbiol, 1983, 46(5): 1146-1151.
[30] Cohen ND, McGruder ED, Neibergs HL, Behle RW, Wallis DE, Hargis BM. Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella[J]. Poult Sci, 1994, 73(2): 354-357.
[31] O'Regan E, McCabe E, Burgess C, McGuinness S, Barry T, Duffy G, Whyte P, Fanning S. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples[J]. BMC Microbiol, 2008, 8:156.
[32] Malorny B, Bunge C, Helmuth R. A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs[J]. J Microbiol Methods, 2007, 70(2): 245-251.
[33] Chen J, Zhang L, Paoli GC, Shi C, Tu SI, Shi X. A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis[J]. Int J Food Microbiol, 2010, 137(2-3): 168-174.
[34] KrascsenicsováK, PiknováL, KaclíkováE, Kuchta T. Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction[J]. Lett Appl Microbiol, 2008, 46(4): 483-487.
[35] Trafny EA, Koz?owska K, Szpakowska M. A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces[J]. Lett Appl Microbiol, 2006, 43(6): 673-679.
[36] Saroj SD, Shashidhar R, Karani M, Bandekar JR. Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture - nested PCR combination assay[J]. Mol Cell Probes, 2008, 22(3): 201-206.
[37] Bansal NS, Gray V, McDonell F. Validated PCR assay for the routine detection of Salmonella in food[J]. J Food Prot, 2006, 69(2): 282-287.
[38] Kawasaki S, Fratamico PM, Horikoshi N, Okada Y, Takeshita K, Sameshima T, Kawamoto S. Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing[J]. Foodborne Pathog Dis, 2009, 6(1): 81-89.
[39] Woo PC, Leung PK, Leung KW, Yuen KY. Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient[J]. Mol Pathol, 2000, 53(4): 211-215.
[40] Ronaghi M, Karamohamed S, Pettersson B, Uhlén M, Nyrén P. Real-time DNA sequencing using detection of pyrophosphate release[J]. Anal Biochem, 1996, 242(1): 84-89.
[41] Agah A, Aghajan M, Mashayekhi F, Amini S, Davis RW, Plummer JD, Ronaghi M, Griffin PB. A multi-enzyme model for Pyrosequencing[J]. Nucleic acids research, 2004, 32(21): 166.
[42] Elahi E, Ronaghi M. Pyrosequencing: a tool for DNA sequencing analysis[J].Methods in molecular biology, 2004, 255(1): 211-219.
[43] Nyrén P, Lundin A. Enzymatic method for continuos monitoring of inorganic pyrophosphate synthesis[J]. Analyt Biochem, 1985, 151(2): 504-509.
[44] Nyrén P. Enzymatic method for continuous monitoring of DNA polymerase activity[J]. Anal Biochem, 1987, 167(2): 235-238.
[45] Nyrén P, Karamohamed S, Ronaghi M. Detection of single-base changes using a bioluminometric primer extension assay[J]. Anal Biochem, 1997, 244(2): 367-373.
[46] Hyman E D. A new method of sequencing DNA[J]. Analyt Biochem, 1988, 174(2): 423-436.
[47] Ronaghi M, Uhlén M, Nyrén P. A sequencing method based on real-time pyrophosphate[J]. Science, 1998, 281(5375): 363-365.
[48] Ronaghi M, Elahi E. Discovery of single nucleotid polymorphisms and mutations by pyrosequencing[J]. Comp Funct Genomics, 2002, 3(1): 51-56.
[49] Langaee T, Ronaghi M. Genetic variation analyses by Pyrosequencing[J]. Mutat Res, 2005, 573(1-2): 96-102.
[50] Klenow H, Overgaard-Hansen K, Patkar SA. Proteolytic cleavage fonative DNA polymerase into two different catalytic fragments.Influence of assay condtions on the change of exonuclease activity and polymerase activity accompanying cleavage[J]. Eur J Biochem, 1971, 22(3): 371-381.
[51] Segel IH, Renosto F, Seubert PA. Sulfate-activating enzymes[J]. Methods Enzymol, 1987, 143:334-349.
[52] Deluca M. Firefly luciferase[J]. Adv Enzymol Relat Areas Mol Biol, 1976, 44:37-68.
[53] Komoszynski M, Wojtczak A. Apyrases (ATP diphosphohydrolases, EC 3.6.1.5): function and relationship to ATPases[J]. Biochim Biophys Acta, 1996, 1310: 233-241.
[54] Ahmadian A, Ehn M, Hober S. Pyrosequencing: History, biochemistry and future[J]. Clin Chim Acta, 2006, 363(1-2): 83-94.
[55] James D Watson. The Human Genome Project: Past, Present, and Future[J]. Science, 1990, 248(4951): 44-49.
[56] David R Bentley. The Human Genome Project-An Overview[J]. Medicinal Research Reviews, 2000, 20(3): 189-196.
[57] Gharizadeh B, Herman ZS, Eason RG, Jejelowo O, Pourmand N. Large-scale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing[J]. Electrophoresis, 2006, 27(15): 3042-3047.
[58] Ronaghi M. Pyrosequencing sheds light on DNA sequencing[J]. Genome Res, 2001, 11(1): 3-11.
[59] Gharizadeh B, Nordstr?m T, Ahmadian A, Ronaghi M, Nyrén P. Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate)Sp-isomer[J]. Anal Biochem, 2002, 301(1): 82-90.
[60] Chen DC, Saarela J, Nuotio I, Jokiaho A, Peltonen L, Palotie A. Comparison of GenFlex Tag array and pyrosequencing in SNP genotyping[J]. J. Mol. Diagn, 2003, 5(4): 243-249.
[61] Lavebratt C, Sengul S. Single nucleotide polymorphism (SNP) allele frequency estimation in DNA pools using Pyrosequencing[J]. Nat Protoc, 2006, 1(6): 2573-2582.
[62] Royo JL, Galán JJ. Pyrosequencing for SNP genotyping[J]. Methods Mol Biol, 2009, 578(3): 123-133.
[63] Lin YS, Liu FG, Wang TY, Pan CT, Chang WT, Li WH. A simple method using PyrosequencingTM to identify de novo SNPs in pooled DNA samples[J]. Nucleic Acids Res, 2010, 3:1-11.
[64] Narasimhan S, Falkenberg VR, Khin MM, Rajeevan MS. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing[J]. BMC Res Notes, 2009, 12(2): 104-112.
[65] Tost J, Gut IG. DNA methylation analysis by pyrosequencing[J]. Nat Protoc, 2007, 2(9): 2265-2275.
[66] Nordstr?m T, Gharizadeh B, Pourmand N, Nyren P, Ronaghi M. Method enabling fast partial sequencing of cDNA clones[J]. Anal Biochem, 2001, 292(2): 266-271.
[67] Garcia CA, Ahmadian A, Gharizadeh B, Lundeberg J, Ronaghi M, Nyrén P. Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene[J]. Gene, 2000, 253(2): 249-257.
[68] Sinclair A, Arnold C, Woodford N. Rapid detection and estimation by pyrosequencing of 23S rRNA genes with a single nucleotide polymorphism conferring linezolid resistance in Enterococci[J]. Antimicrob Agents Chemother[J]. 2003, 47(11): 3620-3622.
[69] Divne AM, Edlund H, Allen M. Forensic analysis of autosomal STR markers using Pyrosequencing [J]. Forensic Sci Int Genet, 2010, 4(2): 122-129.
[70]袁建林,武国军,薛丽,赵锦荣,白玉杰,杨芳,王禾,张运涛,杨力军.应用焦磷酸微测序技术行HLA-DRB基因型分析[J].中国现代医学杂志,2006,16(8):1164-1170.
[71]杨会勇,那广水,朱术会,邹秉杰,奚涛,周国华.利用线性指数PCR-焦磷酸测序技术快速测3种海洋弧菌[J].中国药科大学学报,2009,40(5):460-46.
[72]孟成艳,金嘉琳,雷建强,王莹,张舒,张文宏. PCR-焦磷酸测序法快速检测和鉴定临床常见致病菌的研究[J].中国医学检验杂志,2007,30(4):456-458.
[73]毛静月.焦磷酸测序技术在高危型HpV分型中应用的研[D].第二军医大学硕士学位论文,2008,6.
[74]姚蓝.乙型肝炎病毒Pre-C/BCP突变热点的焦磷酸测序方法的建立及其可靠性研究[D].中山大学硕士学位论文,2007,5.
[75]李秀娟,徐保红,宋红梅,李丽婕,田会芳.沙门菌PCR-焦磷酸测序法检测[J].中国公共卫生,2009,25(8):997-998.
[76]田会方,徐保红,李秀娟,李波,王晓丽. PCR-焦磷酸测序技术在沙门菌食物中毒中的应用[J].中国卫生检验杂志,2009,19(11):2584-2585.
[77] Pai R, Limor J, Beall B. Use of pyrosequencing to differentiate Streptococcus pneumoniae serotypes 6A and 6B[J]. J Clin Microbiol, 2005, 43(9): 4820-4822.
[78] Unnerstad H, Ericsson H, Alderborn A, Tham W, Danielsson-Tham ML, Mattsson JG. Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene[J]. Appl Environ Microbiol, 2001, 67(11): 5339-5342.
[79] Gharizadeh B, Norberg E, L?ffler J, Jalal S, Tollemar J, Einsele H, Klingspor L, Nyrén P. Identification of medically important fungi by the Pyrosequencing technology[J]. Mycoses, 2004 ,47(1-2): 29-33.
[80] Monstein H, Nikpour-Badr S, Jonasson J. Rapid molecular identification and subtyping of Helicobacter pylori by pyrosequencing of the 16S rDNA variable V1 and V3 regions[J]. FEMS Microbiol Lett, 2001, 199(1): 103-107.
[81] Gharizadeh B, Kalantari M, Garcia CA, Johansson B, Nyrén P. Typing of human papillomavirus by pyrosequencing[J]. Lab Invest, 2001, 81(5): 673-679.
[82] Elahi E, Pourmand N, Chaung R, Rofoogaran A, Boisver J, Samimi-Rad K, Davis RW, Ronaghi M. Determination of hepatitis C virus genotype by Pyrosequencing[J]. J Virol Methods, 2003, 109(2): 171-176.
[83] Wahab T, Hjalmarsson S, Wollin R, Engstrand L. Pyrosequencing Bacillus anthracis[J]. Emerg Infect Dis, 2005, 11(10): 1527-1531.
[84] Kobayashi H, Hall GS, Tuohy MJ, Knothe U, Procop GW, Bauer TW. Bilateral periprosthetic joint infection caused by Salmonella enterica serotype Enteritidis, and identification of Salmonella sp using molecular techniques[J]. Int J Infect Dis, 2009, 13(6): e463-e466.
[85] Hebert PD, Ratnasingham S, deWaard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species[J]. Proc Biol Sci, 2003, 270(1): 96-99.
[86] Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH. Use of DNA barcodes to identify flowering plants[J]. Proc Natl Acad Sci USA, 2005, 102(23): 8369-8374.
[87] Taberlet P, Coissac E, Pompanon F, Gielly L, Miquel C, Valentini A, Vermat T, Corthier G, Brochmann C, Willerslev E. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding[J]. Nucleic Acids Res, 2007, 35(3): e14.
[88] Schindel DE, Miller SE. DNA barcoding a useful tool for taxonomists[J]. Nature, 2005, 435(7038): 17.
[89] Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes[J]. Proc Biol Sci, 2003, 270(1512): 313-321.
[90] Waugh J. DNA barcoding in animal species: progress, potential and pitfalls[J]. Bioessays, 2007, 29(2): 188-197.
[91] Aliabadian M, Kaboli M, Nijman V, Vences M. Molecular identification of birds: performance of distance-based DNA barcoding in three genes to delimit parapatric species[J]. PLoS One, 2009, 4(1): e4119.
[92] CBOL Plant Working Group. A DNA barcode for land plants[J]. Proc Natl Acad Sci USA, 2009, 106(31): 12794-12797.
[93] Kim HJ, Park SH, Lee TH, Nahm BH, Chung YH, Seo KH, Kim HY. Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars[J]. J Food Prot, 2006, 69(7): 1653-1661.
[94] Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galán JE, Ginocchio C, Curtiss R 3rd, Gyles CL. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J]. Molecular and Cellular Probes, 1992, 6(4): 271-279.
[95] Bülte M, Jakob P. The use of a PCR-generated invA probe for the detection of Salmonella spp. in artificially and naturally contaminated foods[J]. Int J Food Microbiol, 1995, 26(3): 335-344.
[96] JeníkováG, PazlarováJ, DemnerováK. Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay[J]. Int Microbiol, 2000, 3(4): 225-229.
[97] Chiu CH, Ou JT. Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay[J]. J Clin Microbiol, 1996, 34(10): 2619-2622.
[98] Cocolin L, Manzano M, Cantoni C, Comi G. Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food[J]. J Appl Microbiol, 1998, 85(4): 673-677.
[99] B?umler AJ, Tsolis RM, van der Velden AW, Stojiljkovic I, Anic S, Heffron F. Identification of a new iron regulated locus of Salmonella typhi[J]. Gene, 1996, 183(1-2): 207-213.
[100] B?umler AJ, Heffron F, Reissbrodt R. Rapid detection of Salmonella enterica with primers specific for iroB[J]. J Clin Microbiol, 1997, 35(5): 1224-1230.
[101] Jones DD, Law R, Bej AK. Detection of Salmonella spp. in oysters using polymerase chain reaction (PCR) and gene probes[J]. J. Food Sci, 1993, 58: 1191-1197.
[102] Higgins CF, Haag PD, Nikaido K, Ardeshir F, Garcia G, Ames GF. Complete nucleotide sequence and identification of membrane components of the histidine transport operon of S. typhimurium[J]. Nature, 1982, 298(5876): 723-727.
[103] Mills DM, Bajaj V, Lee CA. A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome[J]. Mol Microbiol, 1995, 15(4): 749-759.
[104] Galán JE. Molecular genetic bases of Salmonella entry into host cells[J]. Mol Microbiol, 1996, 20(2): 263-271.
[105] Bajaj V, Hwang C, Lee CA. hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes[J]. Mol Microbiol, 1995, 18(4): 715-727.
[106] Duguid JP, Smith IW, Dempster G, Edmunds PN. Non-flagellar filamentous appendages (fimbriae) and haemagglutinating activity in Bacterium coli[J]. J Pathol Bacteriol, 1955, 70(2): 335-348.
[107] Clegg S, Swenson DL. Fimbriae: Adhesion, Genetics, Biogenesis, and Vaccines. CRC Press, BocaRaton, FL, In: Klemm, P. (Ed.), 1994, 105-113.
[108] Yeh KS, Chen TH, Liao CW, Chang CS, Lo HC. PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species[J]. Int J Food Microbiol, 2002, 78(3):227-234.
[109] Anonymous. Microbiology of Food and Animal Feeding Stuffs Protocol for the Validation of Alternative Methods (ISO16140:2003). European Committee for Standardization, 2003.
[110] Malorny B, Hoorfar J, Bunge C, Helmuth R. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard[J]. Appl Environ Microbiol, 2003, 69(1): 290-296.