- Introduction
- Materials and Methods
- Bacterial strains and culture conditions.
- Preparation of stool samples inoculated with E. coli strains.
- Clinical stool samples.
- DNA extraction from E. coli strains.
- DNA extraction from stool samples.
- Primer design.
- Conventional PCR assay conditions.
- qPCR assay conditions.
- Plasmid copy number determination.
- DNA hybridization assay.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Hill DR, Beeching NJ, 2010. Travelers' diarrhea. Curr Opin Infect Dis 23: 481–487.
-
2.
Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H, 2010. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect 12: 89–98.
-
3.
Gyles C, So M, Falkow S, 1974. The enterotoxin plasmids of Escherichia coli. J Infect Dis 130: 40–49.
-
4.
Evans DJ Jr, Evans DG, 1973. Three characteristics associated with enterotoxigenic Escherichia coli isolated from man. Infect Immun 8: 322–328.
-
5.
Crossman LC, Chaudhuri RR, Beatson SA, Wells TJ, Desvaux M, Cunningham AF, Petty NK, Mahon V, Brinkley C, Hobman JL, Savarino SJ, Turner SM, Pallen MJ, Penn CW, Parkhill J, Turner AK, Johnson TJ, Thomson NR, Smith SG, Henderson IR, 2010. A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407. J Bacteriol 192: 5822–5831.
-
6.
Kimata K, Shima T, Shimizu M, Tanaka D, Isobe J, Gyobu Y, Watahiki M, Nagai Y, 2005. Rapid categorization of pathogenic Escherichia coli by multiplex PCR. Microbiol Immunol 49: 485–492.
-
7.
Hegde A, Ballal M, Shenoy S, 2012. Detection of diarrheagenic Escherichia coli by multiplex PCR. Indian J Med Microbiol 30: 279–284.
-
8.
Tobias J, Vutukuru SR, 2012. Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli. Microbiol Res 167: 564–570.
-
9.
Fialho OB, de Souza EM, de Borba Dallagassa C, de Oliveira Pedrosa F, Klassen G, Irino K, Paludo KS, de Assis FE, Surek M, de Souza Santos Farah SM, Fadel-Picheth CM, 2013. Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol. J Clin Lab Anal 27: 155–161.
-
10.
Fujioka M, Otomo Y, Ahsan CR, 2013. A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli. J Microbiol Methods 92: 289–292.
-
11.
Liu J, Gratz J, Amour C, Kibiki G, Becker S, Janaki L, Verweij JJ, Taniuchi M, Sobuz SU, Haque R, Haverstick DM, Houpt ER, 2013. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 51: 472–480.
-
12.
Reischl U, Youssef MT, Wolf H, Hyytia-Trees E, Strockbine NA, 2004. Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli. J Clin Microbiol 42: 4092–4100.
-
13.
Bischoff C, Luthy J, Altwegg M, Baggi F, 2005. Rapid detection of diarrheagenic E. coli by real-time PCR. J Microbiol Methods 61: 335–341.
-
14.
Patel CB, Vajpayee P, Singh G, Upadhyay RS, Shanker R, 2011. Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free detection and quantification. Ecotoxicol Environ Saf 74: 2292–2298.
-
15.
Lothigius A, Janzon A, Begum Y, Sjoling A, Qadri F, Svennerholm AM, Bolin I, 2008. Enterotoxigenic Escherichia coli is detectable in water samples from an endemic area by real-time PCR. J Appl Microbiol 104: 1128–1136.
-
16.
Fukushima H, Tsunomori Y, Seki R, 2003. Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools. J Clin Microbiol 41: 5134–5146.
-
17.
Murray BE, Mathewson JJ, DuPont HL, Hill WE, 1987. Utility of oligodeoxyribonucleotide probes for detecting enterotoxigenic Escherichia coli. J Infect Dis 155: 809–811.
-
18.
Hill WE, Payne WL, Zon G, Moseley SL, 1985. Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization. Appl Environ Microbiol 50: 1187–1191.
-
19.
Aagaard K, Petrosino J, Keitel W, Watson M, Katancik J, Garcia N, Patel S, Cutting M, Madden T, Hamilton H, Harris E, Gevers D, Simone G, McInnes P, Versalovic J, 2013. The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters. FASEB J 27: 1012–1022.
-
20.
Jiang ZD, Lowe B, Verenkar MP, Ashley D, Steffen R, Tornieporth N, von Sonnenburg F, Waiyaki P, DuPont HL, 2002. Prevalence of enteric pathogens among international travelers with diarrhea acquired in Kenya (Mombasa), India (Goa), or Jamaica (Montego Bay). J Infect Dis 185: 497–502.
-
21.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG, 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
-
22.
Rozen S, Skaletsky H, 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386.
-
23.
Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO, Sailor CA, Dawson RB, Peek AS, 2008. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36: W163–169.
-
24.
Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S, 2011. Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers. J Microbiol Methods 86: 119–120.
-
25.
Gomez-Duarte OG, Bai J, Newell E, 2009. Detection of Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by 3-reaction multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 63: 1–9.
-
26.
Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methe B, DeSantis TZ, Petrosino JF, Knight R, Birren BW, 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21: 494–504.
-
27.
Hill WE, Wentz BA, Payne WL, Jagow JA, Zon G, 1986. DNA colony hybridization method using synthetic oligonucleotides to detect enterotoxigenic Escherichia coli: collaborative study. J Assoc Off Anal Chem 69: 531–536.
-
28.
Spicer EK, Kavanaugh WM, Dallas WS, Falkow S, Konigsberg WH, Schafer DE, 1981. Sequence homologies between A subunits of Escherichia coli and Vibrio cholerae enterotoxins. Proc Natl Acad Sci USA 78: 50–54.
-
29.
Pecoraro V, Zerulla K, Lange C, Soppa J, 2011. Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-)oligoploid and polyploid species. PLoS ONE 6: e16392.
-
30.
DuPont HL, Formal SB, Hornick RB, Snyder MJ, Libonati JP, Sheahan DG, LaBrec EH, Kalas JP, 1971. Pathogenesis of Escherichia coli diarrhea. N Engl J Med 285: 1–9.
-
31.
Harro C, Chakraborty S, Feller A, DeNearing B, Cage A, Ram M, Lundgren A, Svennerholm AM, Bourgeois AL, Walker RI, Sack DA, 2011. Refinement of a human challenge model for evaluation of enterotoxigenic Escherichia coli vaccines. Clin Vaccine Immunol 18: 1719–1727.
-
32.
Pickering LK, DuPont HL, Evans DG, Evans DJ Jr, Olarte J, 1977. Isolation of enteric pathogens from asymptomatic students from the United States and Latin America. J Infect Dis 135: 1003–1005.
-
33.
So M, Heffron F, McCarthy BJ, 1979. The E. coli gene encoding heat stable toxin is a bacterial transposon flanked by inverted repeats of IS1. Nature 277: 453–456.
-
34.
Nordstrom K, 2006. Plasmid R1–replication and its control. Plasmid 55: 1–26.
-
35.
Womble DD, Dong X, Luckow VA, Wu RP, Rownd RH, 1985. Analysis of the individual regulatory components of the IncFII plasmid replication control system. J Bacteriol 161: 534–543.
-
36.
Zuker M, 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406–3415.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1613 | 1387 | 57 |
| Full Text Views | 492 | 19 | 0 |
| PDF Downloads | 214 | 20 | 0 |
Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples
Bonnie P. Youmans
Bonnie P. Youmans
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Alkek Center for Metagenomics and Microbiome Research, Houston, Texas; Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, Texas; Department of Medicine, Baylor College of Medicine, Division of Infectious Diseases, University of Texas Medical School, St. Luke's Episcopal Hospital, Houston, Texas
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Nadim J. Ajami
Nadim J. Ajami
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Alkek Center for Metagenomics and Microbiome Research, Houston, Texas; Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, Texas; Department of Medicine, Baylor College of Medicine, Division of Infectious Diseases, University of Texas Medical School, St. Luke's Episcopal Hospital, Houston, Texas
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Zhi-Dong Jiang
Zhi-Dong Jiang
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Alkek Center for Metagenomics and Microbiome Research, Houston, Texas; Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, Texas; Department of Medicine, Baylor College of Medicine, Division of Infectious Diseases, University of Texas Medical School, St. Luke's Episcopal Hospital, Houston, Texas
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Joseph F. Petrosino
Joseph F. Petrosino
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Alkek Center for Metagenomics and Microbiome Research, Houston, Texas; Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, Texas; Department of Medicine, Baylor College of Medicine, Division of Infectious Diseases, University of Texas Medical School, St. Luke's Episcopal Hospital, Houston, Texas
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Herbert L. DuPont
Herbert L. DuPont
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Alkek Center for Metagenomics and Microbiome Research, Houston, Texas; Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, Texas; Department of Medicine, Baylor College of Medicine, Division of Infectious Diseases, University of Texas Medical School, St. Luke's Episcopal Hospital, Houston, Texas
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Sarah K. Highlander
Sarah K. Highlander
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Alkek Center for Metagenomics and Microbiome Research, Houston, Texas; Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, Texas; Department of Medicine, Baylor College of Medicine, Division of Infectious Diseases, University of Texas Medical School, St. Luke's Episcopal Hospital, Houston, Texas
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Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.
Author Notes
Financial support: BPY was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases by award no. F31DK094596.
Authors' addresses: Bonnie P. Youmans and Sarah K. Highlander, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, E-mails: youmans@bcm.edu and sarahh@bcm.edu. Nadim J. Ajami and Joseph F. Petrosino, Alkek Center for Metagenomics and Microbiome Research, Baylor College of Medicine, Houston, TX, E-mails: Nadim.Ajami@bcm.edu and jpetrosi@bcm.edu. Zhi-Dong Jiang and Herbert L. DuPont, UT Health, School of Public Health, Houston, TX, E-mails: Zhi-Dong.Jiang@uth.tmc.edu and hdupont@StLukesHealth.org.
- Introduction
- Materials and Methods
- Bacterial strains and culture conditions.
- Preparation of stool samples inoculated with E. coli strains.
- Clinical stool samples.
- DNA extraction from E. coli strains.
- DNA extraction from stool samples.
- Primer design.
- Conventional PCR assay conditions.
- qPCR assay conditions.
- Plasmid copy number determination.
- DNA hybridization assay.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Hill DR, Beeching NJ, 2010. Travelers' diarrhea. Curr Opin Infect Dis 23: 481–487.
-
2.
Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H, 2010. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect 12: 89–98.
-
3.
Gyles C, So M, Falkow S, 1974. The enterotoxin plasmids of Escherichia coli. J Infect Dis 130: 40–49.
-
4.
Evans DJ Jr, Evans DG, 1973. Three characteristics associated with enterotoxigenic Escherichia coli isolated from man. Infect Immun 8: 322–328.
-
5.
Crossman LC, Chaudhuri RR, Beatson SA, Wells TJ, Desvaux M, Cunningham AF, Petty NK, Mahon V, Brinkley C, Hobman JL, Savarino SJ, Turner SM, Pallen MJ, Penn CW, Parkhill J, Turner AK, Johnson TJ, Thomson NR, Smith SG, Henderson IR, 2010. A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407. J Bacteriol 192: 5822–5831.
-
6.
Kimata K, Shima T, Shimizu M, Tanaka D, Isobe J, Gyobu Y, Watahiki M, Nagai Y, 2005. Rapid categorization of pathogenic Escherichia coli by multiplex PCR. Microbiol Immunol 49: 485–492.
-
7.
Hegde A, Ballal M, Shenoy S, 2012. Detection of diarrheagenic Escherichia coli by multiplex PCR. Indian J Med Microbiol 30: 279–284.
-
8.
Tobias J, Vutukuru SR, 2012. Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli. Microbiol Res 167: 564–570.
-
9.
Fialho OB, de Souza EM, de Borba Dallagassa C, de Oliveira Pedrosa F, Klassen G, Irino K, Paludo KS, de Assis FE, Surek M, de Souza Santos Farah SM, Fadel-Picheth CM, 2013. Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol. J Clin Lab Anal 27: 155–161.
-
10.
Fujioka M, Otomo Y, Ahsan CR, 2013. A novel single-step multiplex polymerase chain reaction assay for the detection of diarrheagenic Escherichia coli. J Microbiol Methods 92: 289–292.
-
11.
Liu J, Gratz J, Amour C, Kibiki G, Becker S, Janaki L, Verweij JJ, Taniuchi M, Sobuz SU, Haque R, Haverstick DM, Houpt ER, 2013. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 51: 472–480.
-
12.
Reischl U, Youssef MT, Wolf H, Hyytia-Trees E, Strockbine NA, 2004. Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli. J Clin Microbiol 42: 4092–4100.
-
13.
Bischoff C, Luthy J, Altwegg M, Baggi F, 2005. Rapid detection of diarrheagenic E. coli by real-time PCR. J Microbiol Methods 61: 335–341.
-
14.
Patel CB, Vajpayee P, Singh G, Upadhyay RS, Shanker R, 2011. Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free detection and quantification. Ecotoxicol Environ Saf 74: 2292–2298.
-
15.
Lothigius A, Janzon A, Begum Y, Sjoling A, Qadri F, Svennerholm AM, Bolin I, 2008. Enterotoxigenic Escherichia coli is detectable in water samples from an endemic area by real-time PCR. J Appl Microbiol 104: 1128–1136.
-
16.
Fukushima H, Tsunomori Y, Seki R, 2003. Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools. J Clin Microbiol 41: 5134–5146.
-
17.
Murray BE, Mathewson JJ, DuPont HL, Hill WE, 1987. Utility of oligodeoxyribonucleotide probes for detecting enterotoxigenic Escherichia coli. J Infect Dis 155: 809–811.
-
18.
Hill WE, Payne WL, Zon G, Moseley SL, 1985. Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization. Appl Environ Microbiol 50: 1187–1191.
-
19.
Aagaard K, Petrosino J, Keitel W, Watson M, Katancik J, Garcia N, Patel S, Cutting M, Madden T, Hamilton H, Harris E, Gevers D, Simone G, McInnes P, Versalovic J, 2013. The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters. FASEB J 27: 1012–1022.
-
20.
Jiang ZD, Lowe B, Verenkar MP, Ashley D, Steffen R, Tornieporth N, von Sonnenburg F, Waiyaki P, DuPont HL, 2002. Prevalence of enteric pathogens among international travelers with diarrhea acquired in Kenya (Mombasa), India (Goa), or Jamaica (Montego Bay). J Infect Dis 185: 497–502.
-
21.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG, 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
-
22.
Rozen S, Skaletsky H, 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386.
-
23.
Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO, Sailor CA, Dawson RB, Peek AS, 2008. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36: W163–169.
-
24.
Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S, 2011. Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers. J Microbiol Methods 86: 119–120.
-
25.
Gomez-Duarte OG, Bai J, Newell E, 2009. Detection of Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by 3-reaction multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 63: 1–9.
-
26.
Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methe B, DeSantis TZ, Petrosino JF, Knight R, Birren BW, 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21: 494–504.
-
27.
Hill WE, Wentz BA, Payne WL, Jagow JA, Zon G, 1986. DNA colony hybridization method using synthetic oligonucleotides to detect enterotoxigenic Escherichia coli: collaborative study. J Assoc Off Anal Chem 69: 531–536.
-
28.
Spicer EK, Kavanaugh WM, Dallas WS, Falkow S, Konigsberg WH, Schafer DE, 1981. Sequence homologies between A subunits of Escherichia coli and Vibrio cholerae enterotoxins. Proc Natl Acad Sci USA 78: 50–54.
-
29.
Pecoraro V, Zerulla K, Lange C, Soppa J, 2011. Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-)oligoploid and polyploid species. PLoS ONE 6: e16392.
-
30.
DuPont HL, Formal SB, Hornick RB, Snyder MJ, Libonati JP, Sheahan DG, LaBrec EH, Kalas JP, 1971. Pathogenesis of Escherichia coli diarrhea. N Engl J Med 285: 1–9.
-
31.
Harro C, Chakraborty S, Feller A, DeNearing B, Cage A, Ram M, Lundgren A, Svennerholm AM, Bourgeois AL, Walker RI, Sack DA, 2011. Refinement of a human challenge model for evaluation of enterotoxigenic Escherichia coli vaccines. Clin Vaccine Immunol 18: 1719–1727.
-
32.
Pickering LK, DuPont HL, Evans DG, Evans DJ Jr, Olarte J, 1977. Isolation of enteric pathogens from asymptomatic students from the United States and Latin America. J Infect Dis 135: 1003–1005.
-
33.
So M, Heffron F, McCarthy BJ, 1979. The E. coli gene encoding heat stable toxin is a bacterial transposon flanked by inverted repeats of IS1. Nature 277: 453–456.
-
34.
Nordstrom K, 2006. Plasmid R1–replication and its control. Plasmid 55: 1–26.
-
35.
Womble DD, Dong X, Luckow VA, Wu RP, Rownd RH, 1985. Analysis of the individual regulatory components of the IncFII plasmid replication control system. J Bacteriol 161: 534–543.
-
36.
Zuker M, 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406–3415.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1613 | 1387 | 57 |
| Full Text Views | 492 | 19 | 0 |
| PDF Downloads | 214 | 20 | 0 |