Letters in Applied Microbiology 2001, 32, 126±130
Optimization of random ampli®cation of polymorphic DNA analysis for molecular subtyping of Escherichia coli O157 K.L. Hopkins and A.C. Hilton1
School of Biosciences, University of Birmingham, Edgbaston and 1School of Life and Health Sciences, Aston University, Birmingham, UK 2000/50: received 30 October 2000 and accepted 21 November 2000
Random ampli®cation of polymorphic DNA (RAPD) analysis using the polymerase chain reaction has proved to be a useful technique in the epidemiological investigation of micro-organisms but may suffer from a lack of reproducibility in poorly optimized protocols. In this study a method of obtaining reproducible genomic ®ngerprints using RAPD analysis of Escherichia coli O157 is described. By systematic optimization of reaction conditions and selection of suitable primers, reproducible and discriminatory pro®les could be obtained from all E. coli O157 strains tested. In addition, two other methods of obtaining reproducible pro®les from E. coli O157 strains without the need to purify genomic DNA are described. K . L . H O P K I N S A N D A . C . H I L T O N . 2001.
INTRODUCTION The discriminatory capacity of many methods used to subtype strains of Escherichia coli causing enterohaemorrhagic disease is restricted to clonal strains that are responsible for the majority of cases of infection, particularly serotype O157:H7. In response, research has been directed towards the development of DNA-based methods that can target the often minimal amount of variation available for strain differentiation. Routine use of random ampli®cation of polymorphic DNA (RAPD) analysis as a subtyping method requires a robust polymerase chain reaction (PCR) protocol as the technique can be sensitive to changes in reaction conditions (Hilton et al. 1997; Khandka et al. 1997). It has been shown that different laboratories may produce variable size ranges of fragments from the same strain if there is a lack of standardization (Penner et al. 1993). Variations in the number and intensity of amplicons derived from a single strain may occur when using different DNA polymerases due to slightly different reaction condition requirements (Meunier and Grimont 1993). In addition, differences in the ramp rates between individual thermal cyclers may also result in changes in the pro®le and poor reproducibility, 1 although this was reduced by improved machines (Meunier and Grimont 1993; Penner et al. 1993). Primers may vary in
Correspondence to: Dr A.C. Hilton, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK (e-mail:
[email protected]).
their ability to discriminate between strains depending on their sequence (particularly at the 3¢ end), % G + C content and length (Akopyanz et al. 1992; Caetano-AnolleÂs et al. 1992; Caetano-AnolleÂs 1993); it is, therefore, recommended that at least two independent primers are used to maximize discriminatory capacity (Louie et al. 1996). The composition of the PCR reaction buffer, in particular the concentration of free magnesium and salts such as potassium chloride, can greatly affect the quality and reproducibility of ampli®cation (Saiki 1989). Random ampli®cation of polymorphic DNA analysis is advantageous as a subtyping technique as it only requires very small amounts of template DNA which need not be double stranded or highly puri®ed (Wang et al. 1993). Template DNA can be isolated from cells after lysis, followed by enzymatic removal of RNA and protein and ®nally precipitation, but this is time consuming. Template may also be created by using whole cell cultures (Madico et al. 1995; Hilton et al. 1997) or by sampling colonies directly from agar plates (Coutinho et al. 1993). In this study a method of obtaining reproducible RAPD analysis pro®les from E. coli O157 is described and three methods of template DNA preparation compared. MATERIALS AND METHODS Bacterial isolates Escherichia coli O157 clinical strains (see Table 1) were 2 received from the PHLS laboratory at Birmingham Heartlands Hospital (West Midlands, UK). ã 2001 The Society for Applied Microbiology
OPTIMIZING RAPD FOR ESCHERICHIA COLI O157
Table 1 Strains of Shiga-like toxin (Slt)-producing Escherichia coli subtyped using random ampli®cation of polymorphic DNA analysis Identi®cation no.
Serotype
Slt type
Phage type
909802 902824 902002 912151 908906 910634 908681 NCTC 12080 NCTC 12079 NCTC 12900
O157 O157 O157 O157 O157 O157 O157 O157:H± O157:H7 O157
Slt1 and 2 negative Slt1 and 2 positive Slt1 and 2 positive Slt2 positive Slt2 positive Slt2 positive Not determined Slt2 positive Slt1 and 2 positive Slt1 and 2 negative
34 8 8 2 2 32 2 4 4 Not determined
Preparation of phenol-extracted template DNA Escherichia coli O157 strains were grown at 37 °C with shaking in 5 ml Luria-Burtani broth for 18±24 h. Genomic DNA was extracted as described previously (Hilton et al. 1996). The DNA was checked for purity and concentration and serial dilutions prepared to 25 ng ll±1; 2 ll of each dilution were used in RAPD analysis as template.
127
Table 2 Buffer matrix of opti-prime kit (Schoettlin et al. 1994) 10 mmol l)1 Tris-HCl
MgCl2
pH pH pH pH pH pH
1á5 3á5 1á5 3á5 1á5 3á5
8á3 8á3 8á8 8á8 9á2 9á2
mmol mmol mmol mmol mmol mmol
l±1 l±1 l±1 l±1 l1 l±1
25 mmol l)1 KCl
75 mmol l)1 KCl
Buffer Buffer Buffer Buffer Buffer Buffer
Buffer Buffer Buffer Buffer Buffer Buffer
1 3 5 7 9 11
2 4 6 8 10 12
in a Progene thermal cycler (Techne, Cambridge, UK) as follows: one cycle of 4á5 min at 94 °C followed by ®ve low stringency cycles of 30 s at 94 °C, 1 min at 22 °C, 2 min at 72 °C and 35 high stringency cycles of 30 s at 94 °C, 30 s at 28 °C, 3 min at 72 °C. When primer 1254 or 1283 was used the high stringency primer annealing temperature was increased to 32 °C. The ampli®cation was concluded with 5 min at 72 °C and the reactions stored at 4 °C until analysis. The ampli®cation products were size separated by electrophoresis in 1á2% agarose and recorded using the IS500 digital imaging system (Flowgen, Kent, UK). RESULTS
Whole cell template in random ampli®cation of polymorphic DNA analysis
Effects of reaction buffer on random ampli®cation of polymorphic DNA analysis
Method 1. Boiled whole cell template was prepared as described by Madico et al. (1995); 0á5 ml standing stationary phase culture was boiled for 10 min, diluted 10-fold and 5 ll used as template.
To maximize the discriminatory capacity of RAPD analysis a suf®cient number of amplicons are required to allow discrimination between unrelated isolates, and recognition of related isolates, but not so many as to make pro®le analysis dif®cult and time consuming. Figure 1 shows ampli®cation pro®les obtained using primer 1254 in which the only variable is the reaction buffer. The effect of KCl concentration can be seen by comparing lanes 1 and 2, 3 and 4, 5 and 6, etc. From this it appears that a KCl concentration of 25 mmol l±1 gives the best ampli®cation and increasing the KCl to 35 mmol l±1 appears to decrease the intensity of the amplicons. Increasing the MgCl2 concentration from 1á5 to 3á5 mmol±1 increased the yield of product (lanes 1 and 3, 9 and 11). Increasing the pH did not appear to have much effect on the pro®les obtained when the MgCl2 and KCl concentrations were optimal (lanes 3 and 11). However, at less than optimal concentrations, pH did affect PCR product yield (lanes 4, 8 and 12). Based on these observations, PCR reaction buffer 3 was selected for use in further reactions.
Method 2. Ethanol-treated whole cell template was prepared as described by Hilton et al. (1997). In brief, 3 ml culture were spun down and then resuspended in 1 ml 100% ethanol. The cells were washed in 1 ml phosphate-buffered saline and ®nally resuspended in 1 ml H2O. Serial 10-fold dilutions were made and 2 ll of each dilution used as template. Random ampli®cation of polymorphic DNA analysis The PCR was carried out in a 25-ll volume containing 2á5 ll 10 ´ PCR buffer (Table 2; Stratagene, La Jolla, CA, 3 USA), 0á5 ll 10 mmol l±1 dNTPs (Bioline, London, UK), 0á6 ll 100 mmol l±1 primer (1247, 1254, 1283 or 1290 (Akopyanz et al. 1992)), 1á25 U Taq polymerase (GibcoBRL, Paisley, UK), 19á15 ll distilled H2O and 2 ll extracted DNA template or 2 ll diluted ethanol-treated whole cell culture. When boiled whole cells were used only 16á15 ll distilled H2O were added. Reactions were ampli®ed
Random ampli®cation of polymorphic DNA analysis using extracted DNA Ampli®cation was performed using reaction buffer 3 and primer 1254 with dilutions of phenol-extracted DNA as
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 126±130
128 K . L . H O P K I N S A N D A . C . H I L T O N
Fig. 1 Random ampli®cation of polymorphic DNA by polymerase chain reaction pro®les obtained from Escherichia coli O157 genomic DNA. Lanes numbered according to the 12 4 buffers detailed in Table 2. M, 1 kb DNA molecular weight ladder (GibcoBRL)
while lower concentrations did not support ampli®cation of some high molecular weight amplicons. Comparison of pro®les generated using phenolextracted DNA and whole cell templates The whole cell template of E. coli O157 was prepared as described in Materials and Methods and used as template in RAPD analysis with primer 1254. The pro®les obtained from boiled whole cell template, ethanol-treated whole cell template diluted to 10±3 and phenol-extracted DNA were virtually identical (Fig. 3, lanes B, E±3 and P). Selection of discriminatory primer(s)
Fig. 2 Random ampli®cation of polymorphic DNA by polymerase chain reaction (PCR) pro®les obtained from diluted genomic DNA of Escherichia coli O157. M, 1 kb DNA molecular weight ladder (GibcoBRL); TC, no template control
template (Fig. 2). DNA (20 ng) added to reactions gave optimal ampli®cation. Higher DNA concentrations showed no improvement in ampli®cation and a higher background,
Random ampli®cation of polymorphic DNA analysis was carried out using primers 1254, 1247, 1283 or 1290 and the pro®les obtained are shown in Fig. 4. All four primers were capable of producing useful pro®les of 12±19 fragments between ca 200 and 3000 bp, although primer 1247 resulted in a lower yield of amplicons than the other three primers (Fig. 4d). Primer 1290 gave the best intraserotype differentiation based on the number of different pro®les obtained (Fig. 4c). Primer 1290 differentiated six different types, compared with three for primer 1283, four for primer 1254 and ®ve for primer 1247. DISCUSSION In this study the PCR was used to amplify randomly primed genomic DNA from E. coli O157 strains using a protocol
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 126±130
OPTIMIZING RAPD FOR ESCHERICHIA COLI O157
M
E–3
B
P
M
bp 4072 3054 2036 1636 1018
517 396
Fig. 3 Comparison of random ampli®cation of polymorphic DNA by polymerase chain reaction pro®les obtained from Escherichia coli O157 genomic DNA template prepared by different methods. Lanes: M, 1 kb DNA molecular weight ladder (GibcoBRL); P, phenol-extracted DNA; E±3, 10±3 dilution ethanol-treated cells; B, boiled cell template
optimized in terms of reaction buffer ionic concentration, template concentration, template preparation method and choice of primer. Figure 1 shows the importance of the correct choice of PCR reaction buffer in RAPD analysis. Cations such as K+ or NH4+ affect PCR speci®city by binding to the negatively charged phosphate groups on DNA, thus facilitating primer±template annealing. The yield and speci®city of the PCR can thereby be increased by altering the concentration of KCl. Magnesium chloride stabilizes primer annealing; therefore, the concentration of MgCl2 can have a large effect on the speci®city and yield of a reaction (Saiki 1989). Too little Mg2+ can decrease the yield whilst excess Mg2+ results in non-speci®c ampli®cation as a result of reduced enzyme ®delity. In contrast to these ®ndings,
129
Meunier and Grimont (1993) produced identical pro®les when three reaction buffers differing in composition were interchanged using the same brand of Taq DNA polymerase. Sensitivity to small changes in pH at certain MgCl2 and KCl concentrations led Hilton et al. (1997) to highlight the importance of accurate pH measurement during reaction buffer preparation. However, the development of commercially available prealiquoted PCR master mixes and single dose reaction beads containing reaction buffer, dNTPs and DNA polymerase may increase reproducibility by removing variations during reaction buffer preparation. The ratio of DNA template to primer is one of the most critical factors to consider when optimizing the PCR; therefore, a range of DNA concentrations should be titred against a ®xed primer concentration for each DNA extraction protocol to obtain the ideal conditions (Tyler et al. 1997). Although it has been suggested that phenol-extracted DNA can aid reproducibility, using whole cell template may save time during investigation of large outbreaks. Figure 3 shows that whole cell template is a good substitute for phenol-extracted DNA. However, comparison of phenolextracted DNA template pro®les and boiled whole cell template pro®les by Madico et al. (1995) revealed that pro®les were usually similar but some differences occurred as a result of damage to large DNA fragments during boiling. This may also lead to a lack of reproducibility as damage to the DNA is a random event. Hilton et al. (1997) found that ethanol-treated whole cell template gave pro®les almost identical to those obtained from phenol-extracted DNA. However, reaction products were less stable and pro®les smeared if not analysed within 24 h post ampli®cation. Alos et al. (1993) suggested that, due to the distribution of binding sites, certain primers may amplify regions of the genome that overlap and, therefore, amplify from only a small section of the genome, thus possibly not re¯ecting the true heterogeneity between strains. Other primers may amplify different parts of the genome and so differentiate between strains, it is, therefore, important to try more than one primer in RAPD analysis. Primers 1290, 1283, 1254 and 1247 have been used previously for successful RAPD analysis of E. coli O157 (Madico et al. 1995) and enterotoxigenic E. coli serotypes (Pacheco et al. 1998). Pacheco et al. (1997) also recommended primer 1290 for detection of intraserotype polymorphism. Nevertheless, the lack of heterogeneity seen between strains of E. coli O157 subtyped using RAPD analysis with each of the four primers re¯ects the clonal nature of the group (Whittam et al. 1988). Researchers are apprehensive about using RAPD analysis for subtyping because of problems with poor reproducibility and sensitivity to reaction conditions. However, systematic optimization of the reaction conditions, which may be aided by the use of commercially available reaction buffer
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 126±130
130 K . L . H O P K I N S A N D A . C . H I L T O N
optimization kits, and careful choice of primer can result in highly discriminatory and reproducible RAPD analysis of E. coli O157. ACKNOWLEDGEMENTS This work was funded by a grant from the Division of Environmental Health and Risk Management, University of Birmingham, UK. Escherichia coli O157 strains were gratefully received from the PHLS laboratory, Heartlands Hospital, West Midlands, UK. REFERENCES Akopyanz, N., Bukanov, N.O., Westblom, T.U., Kresovich, S. and Berg, D.E. (1992) DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD-®ngerprinting. Nucleic Acids Research 20, 5137±5142. Alos, J.I., Lambert, T. and Courvalin, P. (1993) Comparison of two molecular typing methods for tracing nosocomial transmission of Escherichia coli K1 in a neonatal unit. Journal of Clinical Microbiology 31, 1704±1709. Caetano-AnolleÂs, G. (1993) Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods and Applications 3, 85±94. Caetano-AnolleÂs, G., Bassam, B.J. and Gresshof, P.M. (1992) Primer± template interactions during DNA ampli®cation ®ngerprinting with single arbitrary oligonucleotides. Molecular and General Genetics 235, 157±165. Coutinho, H.L.C., Handley, B.A., Kay, H.E., Stevenson, L. and Beringer, J.E. (1993) The effect of colony age on PCR ®ngerprinting. Letters in Applied Microbiology 17, 282±284. Hilton, A.C., Banks, J.G. and Penn, C.W. (1996) Random ampli®cation of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of ampli®ed sequences. Journal of Applied Bacteriology 81, 575±584. Hilton, A.C., Banks, J.F. and Penn, C.W. (1997) Optimization of RAPD for ®ngerprinting Salmonella. Letters in Applied Microbiology 24, 243±248. Khandka, D.K., Tuna, M., Tal, M., Nejidat, A. and Golan-Goldhirsh, A. (1997) Variability in the pattern of random ampli®ed polymorphic DNA. Electrophoresis 18, 2852±2856. Louie, M., Jayaratne, P., Luchsinger, I., Devenish, J., Yao, J., Schlech, W. and Simor, A. (1996) Comparison of ribotyping, arbitrarily
primed PCR, and pulsed-®eld gel electrophoresis for molecular typing of Listeria monocytogenes. Journal of Clinical Microbiology 34, 15±19. Madico, G., Akopyants, N.S. and Berg, D.E. (1995) Arbitrarily primed PCR DNA ®ngerprinting of Escherichia coli O157:H7 strains by using templates from boiled cultures. Journal of Clinical Microbiology 33, 1534±1536. Meunier, J.-R. and Grimont, P.A.D. (1993) Factors affecting reproducibility of random ampli®ed polymorphic DNA ®ngerprinting. Research in Microbiology 144, 373±379. Pacheco, A.B.F., Guth, B.E.C., Soares, K.C.C., Nishimura, L., de Almeida, D.F. and Ferreira, L.C.S. (1997) Random ampli®cation of polymorphic DNA reveals serotype-speci®c clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans. Journal of Clinical Microbiology 35, 1521±1525. Pacheco, A.B.F., Soares, K.C., de Almeida, D.F., Viboud, G.I., Binsztein, N. and Ferreira, L.C.S. (1998) Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly ampli®ed polymorphic DNA analysis. Journal of Clinical Microbiology 36, 2099±2102. Penner, G.A., Bush, A., Wise, R., Kim, W., Domier, L., Kasha, K., Laroche, A., Scoles, G., Molnar, S.J. and Fedak, G. (1993) Reproducibility of random ampli®ed polymorphic DNA (RAPD) analysis among laboratories. PCR Methods and Applications 2, 341±345. Saiki, R. (1989) The design and optimisation of the PCR. In PCR Technology: Principles and Applications for DNA Ampli®cation ed. Erlich, A. pp. 7±16. New York: Macmillan. Schoettlin, W., Nielson, K.B. and Mathur, E. (1994) Optimisation of PCR using the opti-prime kit. Strategies in Molecular Biology Newsletter 6, 43±44. Tyler, K.D., Wang, G., Tyler, S.D. and Johnson, W.M. (1997) Factors affecting reliability and reproducibility of ampli®cation-based DNA ®ngerprinting of representative bacterial pathogens. Journal of Clinical Microbiology 35, 339±346. Wang, G., Whittam, T.S., Berg, C.M. and Berg, D.E. (1993) RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains. Nucleic Acids Research 21, 5930±5933. Whittam, T.S., Wachsmuth, I.K. and Wilson, R.A. (1988) Genetic evidence of clonal descent of Escherichia coli O157:H7 associated with hemorrhagic colitis and hemolytic uremic syndrome. Journal of Infectious Diseases 157, 1124±1133.
ã 2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 126±130
