Advanced
Development of a One-Step PCR Assay with Nine Primer Pairs for the Detection of Five Diarrheagenic Escherichia coli Types
Development of a One-Step PCR Assay with Nine Primer Pairs for the Detection of Five Diarrheagenic Escherichia coli Types
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 862-868
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : December 12, 2013
  • Accepted : March 13, 2014
  • Published : June 30, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Kyung-Hwan Oh
Division of Enteric Diseases, Center for Infectious Diseases, Korea National Institute of Health, Osong 363-95, Republic of Korea
Soo-Bok Kim
Kogene Biotech Co., Ltd. Geumcheon-gu, Seoul 153-786, Republic of Korea
Mi-Sun Park
Kogene Biotech Co., Ltd. Geumcheon-gu, Seoul 153-786, Republic of Korea
Seung-Hak Cho
Division of Enteric Diseases, Center for Infectious Diseases, Korea National Institute of Health, Osong 363-95, Republic of Korea
skcho38@korea.kr

Abstract
Certain Escherichia coli ( E. coli ) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MPPCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented ( i.e. , stx 1 and stx 2 for EHEC, lt , st h, and st p for ETEC, eae A and bfp A for EPEC, agg R for EAEC, and ipa H for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: 5 × 10 1 CFU/ml for EHEC, 5 × 10 3 CFU/ml for ETEC expressing lt and st h, 5 × 10 4 CFU/ml for ETEC expressing st p, 5 × 10 2 CFU/ml for EPEC, 5 × 10 4 CFU/ml for EAEC, and 5 × 10 2 CFU/ml for EIEC. To confirm the specificity, C. jejuni , C. perfringens , S. Typhimurium, V. parahaemolyticus , L. monocytogenes , Y. enterocolitica , B. cereus , and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli .
Keywords
Introduction
Diarrheal diseases are extraordinarily common with a worldwide distribution, and diarrheagenic Escherichia coli ( E. coli ) strains are important causative agents [3] .
Five types of diarrheagenic E. coli have been identified, namely enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [11] . The major virulence factor, which is also a defining characteristic of EHEC, is Shiga toxin (Stx). Shiga toxin-producing E. coli (STEC) strains produce one or both of the two major types of Shiga toxins, designated Stx1 and Stx2. Production of Stx2 is associated with an increased risk of developing the hemolytic-uremic syndrome [20] . Sequence homologies for the A and B subunits are 55% and 57% for the prototypical Stx1 and Stx2 toxins, respectively [11 , 13] . ETEC strains are identified by the ability to produce enterotoxins; the heatlabile toxin (LT) and the heat-stable toxin (ST) [19] . The LT toxin is encoded by elt AB, and the ST toxin is encoded by two different genes, est A and st 1, which produce STh (originally isolated from ETEC in humans) and STp (originally from a pig isolate) [8 , 10 , 11] . EPECs can be classified as typical or atypical based on the production of bundle-forming pili (BFP) encoded by the Escherichia adherence factor (EAF) gene [11] . Atypical EPEC strains would possess the eae gene that correlates with possession of the 35 kb locus of enterocyte effacement (LEE) pathogenicity island responsible for the attaching and effacing (A/E) effect [22] . A 1 kb fragment of the 60-65 MDa virulence plasmid (pAA) has been used for the detection of EAEC. The pAA encodes AA fimbriae (AAF) I, II, and III [1] ; the transcriptional activator AggR [12] ; enteroaggregative heatstable enterotoxin 1 (EAST-1) [16] ; and an antiaggregation protein, dispersin [18] . EIEC strain infections are associated with watery diarrhea and inflammation, with fever resembling that observed during Shigella infection, and are mediated by the invasion-associated plasmid antigen, ipa H [17] .
The multiplex PCR (MP-PCR) assay is a useful method for the simultaneous detection of numerous target genes in a single sample. To detect the five diarrheagenic E. coli types rapidly, a one-step MP-PCR assay with nine primers was developed in this study. The sensitivity and specificity of the primers were tested using diarrheagenic E. coli and non- E. coli enteric bacterial strains. The capacity of detection was validated using 100 fecal samples from patients with diarrhea, and 150 strains of diarrheagenic E. coli isolated from patients in Korea.
Materials and Methods
- Bacterial Strains and Fecal Samples
As shown in Table 1 , reference strains were used for the positive detection of target genes. Campylobacter jejuni , Clostridium perfringens , Salmonella Typhimurium , Vibrio parahaemolyticus , Listeria monocytogenes , Yersinia enterocolitica , Bacillus cereus , and Staphylococcus aureus were used as negative controls. To evaluate the developed MP-PCR assay, we used 100 fecal samples from patients with diarrhea, collected from various provinces in Korea, and 150 strains of diarrheagenic E. coli isolated from patients in Korea ( Tables 2 and 3 ).
E. colireference strains and negative strains used in this study.
PPT Slide
Lager Image
E. coli reference strains and negative strains used in this study.
PathogenicE. coliisolates used in this study.
PPT Slide
Lager Image
Pathogenic E. coli isolates used in this study.
Fecal samples used in this study, grouped by patient origin and time of collection.
PPT Slide
Lager Image
Fecal samples used in this study, grouped by patient origin and time of collection.
- DNA Preparation
Fecal samples were incubated in tryptic soy broth (TSB) at 37℃ in a shaking incubator. After 18 h, the culture medium was centrifuged at 10,000 × g and the supernatant was discarded. The pellet was washed by suspending it in a 0.85% saline solution and centrifuging it. After washing three times, the pellet was resuspended in 1 ml of DW. Reference strains (positive and negative control strains) and diarrheagenic E. coli isolated from patients with diarrhea were incubated in TSB at 37℃ in a shaking incubator.
Total genomic DNA was extracted according to the manufacturer’s instructions using the QIAamp DNA Mini Kit (51306; Qiagen, Germany).
- Construction of PCR Primers
Nine primer pairs were designed based on virulence gene sequences retrieved from the National Center for Biotechnology Information (NCBI) database. The gene primers were constructed using MultAlin ( http://bioinfo.genotoul.fr/multalin/multalin.html ), which uses a matrix constructed by pairwise alignment of scores [4] . The resulting amplicons ranged from 100 to 800 bp in length.
- PCR
For PCR, reaction mixtures that contained 10 μl of 2× DNA polymerase enzyme (PowerAmp 2× premix), 4 μl of primer mixtures, 5 μl of template DNA, and sterile distilled water to bring the final volume to 20 μl were prepared. The PCR was performed using the TaKaRa PCR Thermal Cycler Dice TP600 (TaKaRa, Japan). The reaction was started with a 15 min denaturation step at 95℃. The amplification cycle was as follows: 30 sec at 95℃, followed by 1 min at 58℃ and 1 min at 72℃. Each cycle was repeated 35 times. The final cycle was followed by incubation of the reaction mixture for 10 min at 72℃. The amplified DNA was separated by submarine gel electrophoresis on 1% agarose, stained with ethidium bromide, and visualized under UV transillumination. The PCR experiment was performed in triplicate to verify the constructed PCR primers.
Results
- Development of a Multiplex PCR Assay for the Detection of Five Pathogenic E. coli Types Causing Diarrhea
For the development of an MP-PCR assay to detect diarrheagenic E. coli , nine primer pairs were designed. Primers were selected in order to amplify the sequences of the target genes obtained through GenBank. Highly conserved virulence genes in the representative strains of five E. coli virotypes were used; namely, stx 1 and stx 2 for EHEC; lt , st h, and st p for ETEC; eae A and bfp A for EPEC; agg R for EAEC; and ipa H for EIEC ( Table 1 ). Various combinations of sequences and concentrations of the primers were tested for the optimization ( Table 4 ). The primer pairs were selected to give rise to DNA fragments of different sizes. Amplification with these primers yielded products of expected sizes that could be distinguished by agarose gel electrophoresis ( Fig. 1 ): 141 bp ( ipa H), 160 bp ( st p), 167 bp ( st h), 248 bp ( eae A), 297 bp ( stx 2), 400 bp ( bfp A), 500 bp ( lt ), 637 bp ( stx 1), and 715 bp ( agg R). An MP-PCR containing all nine primer pairs was performed using a mixed DNA sample ( Fig. 1 , lane 12).
Primer pairs used for detection of marker virulence genes indicative of the pathogenicE. colitypes.
PPT Slide
Lager Image
Primer pairs used for detection of marker virulence genes indicative of the pathogenic E. coli types.
PPT Slide
Lager Image
Size comparison of the PCR products. Lane 1: 100 bp DNA ladder, lane 2: negative control, lane 3: ipaH (141 bp), lane 4: stp (160 bp), lane 5: sth (167 bp), lane 6: eaeA (248 bp), lane 7: stx2 (297 bp), lane 8: bfpA (400 bp), lane 9: lt (500 bp), lane 10: stx1 (637 bp), lane 11: aggR (715 bp), lane 12: nine PCR products generated from a mixed DNA sample.
- Specificity and Sensitivity Measurement of the MP-PCR Primers
The constructed MP-PCR primer kit was tested using 25 E. coli reference strains from the five virotypes (EHEC, ETEC, EPEC, EAEC, and EIEC), and eight non- E. coli enteric microorganisms, as shown in Table 1 . Based on the production of amplified PCR products of the expected size for all the strains within a particular virotype, the primers were virotype-specific ( Fig. 2 ). As negative controls, Campylobacter jejuni , Clostridium perfringens , Salmonella Typhimurium, Vibrio parahaemolyticus , Listeria monocytogenes , Yersinia enterocolitica , Bacillus cereus , and Staphylococcus aureus were used to test the primer specificity. No PCRs were obtained for the DNA isolated from the negative controls. To evaluate sensitivity of the MP-PCR primers, the limit of detection (LOD) was measured. The LOD for each primer pair was as follows: 5 × 10 1 CFU/ml for EHEC, 5 × 10 3 CFU/ml for ETEC, 5 × 10 2 CFU/ml for EPEC, 5 × 10 4 CFU/ml for EAEC, and 5 × 10 2 CFU/ml for EIEC ( Fig. 3 ).
PPT Slide
Lager Image
Specificity of MP-PCR against diarrheagenic E. coli strains (A) and non-E. coli strains (B). (A) Lane 1: 100-bp DNA ladder; lane 2: negative control; lane 3: positive control mix; lane 4: EPEC (eaeA); lane 5: EHEC (eaeA and stx2); lane 6: EIEC (ipaH), ETEC (sth) and EHEC (eaeA and stx2); lane 7: ETEC (sth) and EHEC (eaeA and stx2); lane 8: EAEC (aggR), ETEC (sth) and EHEC (eaeA, stx1 and stx2); lane 9: ETEC (lt and sth); lane 10: ETEC (lt and sth); lane 11: ETEC (sth) and EHEC (stx1 and stx2); lane 12: ETEC (lt) and EPEC(eaeA and bfpA); lane 13: EIEC (ipaH); lane 14: EPEC (eaeA and bfpA); lane 15: EAEC (aggR). (B) Lane 1: 100-bp DNA ladder; lane 2: negative control; lane 3: positive control mix; lane 4: Campylobacter jejuni; lane 5: Clostridium perfringens; lane 6: EHEC O157 (stx1 and stx2); lane 7: Salmonella Typhimurium; lane 8: Vibrio parahaemolyticus; lane 9: Listeria monocytogenes; lane 10: Yersinia enterocolitica, lane 11: Bacillus cereus, lane 12: Staphylococcus aureus.
PPT Slide
Lager Image
Sensitivity of MP-PCR as determined by the limits of detection. ETEC (lt and sth) (A), EAEC (aggR) (B), EPEC (eaeA and bfpA) (C), EIEC (ipaH) (D), EHEC (eaeA, stx1, and stx2) (E), and ETEC (stp) (F). Lane 1: 100 bp DNA ladder; lane 2: Negative control; lane 3: Positive control; lane 4: 5 × 108 CFU/ml; lane 5: 5 × 107 CFU/ml; lane 6: 5 × 106 CFU/ml; lane 7: 5 × 105 CFU/ml; lane 8: 5 × 104 CFU/ml; lane 9: 5 × 103 CFU/ml; lane 10: 5 × 102 CFU/ml; lane 11: 5 × 101 CFU/ml.
- Validation of the MP-PCR with Clinical Isolates and Fecal Samples
To validate the developed MP-PCR assay, we analyzed 140 isolates from our strain collection derived from patients with diarrhea from the Chungcheongnam-do, Gyeonggido, Gyeongsangnam-do, and Jeollanam-do provinces between 2003 and 2011, and 10 inflow strains of EIEC isolated from travelers in foreign countries between 2008 and 2011 ( Table 2 ). We also analyzed 100 fecal samples of patients with diarrhea from various regions ( Table 3 ). The analysis revealed 40 EHEC, 40 ETEC, 30 EPEC, 30 EAEC, and 10 EIEC strains that had already been confirmed using PCR assays to amplify sequences specific to individual virulence genes (data no shown). As shown in Fig. 4 , the PCR products amplified using the feces of patients with diarrhea as a starting material were the same as the PCR products amplified using the patient isolates. These results indicated that the multiplex PCR assay could effectively detect the toxin genes of diarrheagenic E. coli ( Table 5 ).
PPT Slide
Lager Image
Products of MP-PCR with feces of diarrheal patients. Lane 1: 100 bp DNA ladder; lane 2: eaeA, stx1, and stx2; lane 3: st; lane 4: st; lane 5: aggR; lane 6: eaeA and bfpA; lane 7 stx1 and stx2; lane 8: aggR, lane 9: aggR; lane 10: lt and st; lane 11: eaeA and bfpA; lane 12: lt and st; lane 13: Positive control; lane 14: Negative control.
Detection of virulence genes of 150E. coliisolates.
PPT Slide
Lager Image
Detection of virulence genes of 150 E. coli isolates.
Discussion
Multiplex PCR has been used to minimize the time and materials required to simultaneously detect multiple target genes in a single sample. In this study, nine specific primer pairs were designed to detect five diarrheagenic E. coli types (EHEC, ETEC, EPEC, EAEC, and EIEC), and an assay that could simultaneously detect all nine different genes in a single MP-PCR was developed. As shown in Table 1 , typical virulence genes were used for each of the five E. coli virotypes.
Nonspecific amplification can often occur because of nonspecific reaction of mixed PCR primers [2 , 9 , 23] ; therefore, optimizing the sensitivity and specificity of this assay is crucial. In this study, nonspecific amplifications were also observed in the initial experiments. However, various primer combinations were tested until nine primer pairs were developed. Finally, the nonaplex-PCR developed in this study successfully amplified mixed DNA of all target genes simultaneously.
Most of the reported MP-PCR methods for detection of pathogenic E. coli have the capacity to amplify several target genes [7 , 14 , 15] . However, these assays applied three or four different MP-PCR reactions. Watterworth et al . [23] developed an MP-PCR assay for the detection of six virulence genes from four categorized diarrheagenic E. coli , and Tobias and Vutukuru [21] developed an mPCR assay for the identification of four categorized diarrheagenic E. coli by the detection of eight virulence genes. Both these assays applied to four categorized diarrheagenic E. coli . In this study, an improved mPCR assay was developed to identify five E. coli virotypes by detecting nine target virulence genes.
Fujioka et al . [5 , 6] reported t he development o f two mPCR assays for the simultaneous detection of nine target genes and developed an improved single-step mPCR assay for the detection of 10 target virulence genes from five categorized diarrheagenic E. coli . The ast A codes for EAST-1, but it is widely distributed in diarrheagenic E. coli , and its role in pathogenicity remains unclear [6] . Therefore, in this study, the mPCR was simplified by excluding ast A from the list of target virulence genes. The novel nonaplex-PCR kit was further evaluated using 100 fecal samples from patients with diarrhea and 150 clinical E. coli strains isolated from patients with diarrhea. The novel nonaplex-PCR kit could detect five different diarrheagenic E. coli virotypes from fecal samples as well as complex culture media. This is a significant attribute, as fecal samples from patients with diarrhea often contain nonpathogenic as well as multiple strains of pathogenic E. coli .
The advantage of this assay is that the nine primer pair combination used detected target genes from five different diarrheagenic E. coli virotypes with no false-negative results. This assay also detected mixed infection by more than two strains of pathogenic E. coli . Moreover, this method reduces assay costs and the time required, as it is a single-step assay. Therefore, this technique for the detection of diarrheagenic E. coli will be useful as a rapid and economical method.
Acknowledgements
This study was supported by grants from the National Institute of Health (NIH 4800-4845-300 and NIH 4800-4847-300 to S.H.C.), Republic of Korea.
References
Bernier C , Gounon P , Le Bouguenec C 2002 Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family. Infect. Immun. 70 4302 - 4311    DOI : 10.1128/IAI.70.8.4302-4311.2002
Botteldoorn N , Heyndrickx M , Rijpens N , Herman L 2003 Detection and characterization of verotoxigenic Escherichia coli by VTEC/EHEC multiplex PCR in porcine faeces and pig carcass swabs. Res. Microbiol. 154 97 - 104    DOI : 10.1016/S0923-2508(03)00028-7
Clarke SC 2001 Diarrhoeagenic Escherichia coli: an emerging problem? Diagn. Microbiol. Infect. Dis. 41 93 - 99    DOI : 10.1016/S0732-8893(01)00303-0
Corpet F 1988 Multiple sequence alignment with hierarchical clustering. Nucl. Acids Res. 16 10881 - 10890    DOI : 10.1093/nar/16.22.10881
Fujuoka M , Saito M , Otomo Y 2008 Direct detection of diarrheagenic Escherichia coli in patient stool specimens by developed multiplex PCRs for the establishment of surveillance system of diarrheagenic Escherichia coli. Jpn. J. Med. Technol. 8 1041 - 1046
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    DOI : 10.1016/j.mimet.2012.12.010
Gomez-Duarte OG , Bai J , Newel E 2009 Detection of E. coli, Salmonella spp., Shigella spp., Yersinia entercolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by threereaction multplex PCR. Diagn. Microbiol. Infect. Dis. 63 1 - 9    DOI : 10.1016/j.diagmicrobio.2008.09.006
Kaper JB , Nataro JP , Mobley HL 2004 Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2 123 - 140    DOI : 10.1038/nrmicro818
Lang AL , Tsai YL , Mayer CL , Patton KC , Palmer CJ 1994 Multiplex PCR for detection of the heat-labile toxin gene and Shiga-like toxin I and II genes in Escherichia coli isolated from natural waters. Appl. Environ. Microbiol. 60 3145 - 3149
Lasaro MA , Rodrigues JF , Mathias-Sanos C , Guth BE , Balan A , Sbrogio-Almeida ME , Ferreira LC 2008 Genetic diversity of heat-labile toxin expressd by enterotoxigenic Escherichia coli strains isolated from humans. J. Bacteriol. 190 2400 - 2410    DOI : 10.1128/JB.00988-07
Nataro JP , Kaper JB 1998 Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11 142 - 201
Nataro JP , Deng Y , Walker K 1994 AggR, a transcriptional activator of aggregative adherence factor I expression. J. Bacteriol. 176 4691 - 4699
Osek J 2003 Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes. J. Appl. Microbiol. 95 1217 - 1225    DOI : 10.1046/j.1365-2672.2003.02091.x
Pass MA , Odedra R , Batt RM 2000 Multiplex PCR for identification of Escherichia coli virulence genes. J. Clin. Microbiol. 38 2001 - 2004
Rappelli P , Maddau G , Mannu F , Colombo MM , Fiori PL , Cappuccinelli P 2001 Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli. Microbiologica 24 77 - 83
Savarino SJ , Fasano A , Watson J , Martin BM , Levine MM , Guandalini S , Guerry P 1993 Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another family of E. coli heat-stable toxin. Proc. Natl. Acad. Sci. USA 90 3093 - 3097    DOI : 10.1073/pnas.90.7.3093
Sethabutr O , Venkatesan M , Yam S , Pang LW , Smoak BL , Sang WK 2000 Detection of PCR products of the ipaH gene from Shigella and enteroinvasive Escherichia coli by enzyme linked immunosorbent assay. Diagn. Microbiol. Infect. Dis. 37 11 - 16    DOI : 10.1016/S0732-8893(00)00122-X
Sheikh J , Czezulin JR , Harrington S , Hicks SI , Henderson R , Bouguenec Cle 2002 A novel dispersin protein in enteroaggregative Escherichia coli. J. Clin. Investig. 110 1329 - 1337    DOI : 10.1172/JCI16172
Sjöling Å , Wiklund G , Savarino SJ , Cohen DI , Svennerholm AM 2007 Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. J. Clin. Microbiol. 45 3295 - 3301    DOI : 10.1128/JCM.00471-07
Tarr PI , Gordon CA , Handler CWL 2005 Shiga-toxinproducing Escherichia coli and haemolytic uraemic syndrome. Lancet 365 1073 - 1086
Tobias J , Vutukuru SR 2012 Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli. Microbiol. Res. 167 564 - 570    DOI : 10.1016/j.micres.2011.11.006
Trabulsi LR , Keller R , Gomes TAT 2002 Typical and atypical enteropathogenic Escherichia coli. Emerg. Infect. Dis. 8 508 - 513    DOI : 10.3201/eid0805.010385
Watterworth L , Topp E , Schraft H , Leung KT 2005 Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli. J. Microbiol. Methods 60 93 - 105    DOI : 10.1016/j.mimet.2004.08.016