Rapid, sensitive, microbial detection by gene amplification using restriction endonuclease target sequences

Molecular and Cellular Probes (1997) 11, 297–308

Rapid, sensitive, microbial detection by gene amplification using restriction endonuclease target sequences Louise A. Metherell, Carolyn Hurst and Ian J. Bruce∗ School of Chemical and Life Sciences, University of Greenwich, London SE18 6PF, UK (Received 22 April 1997, Accepted 16 June 1997) The use of primers synthesized to eight class II restriction endonuclease target sequences, from Haemophilus parainfluenzae, Escherichia coli, Staphylococcus aureus, Salmonella infantis, Rhodobacter sphaeroides, Klebsiella pneumoniae, Bacillus amyloliquefaciens and Proteus vulgaris for single and multiplex PCR identification of the organisms is discussed. Results indicate that the method is sensitive and specific enough to detect single cells and attogram amounts of target DNA. It has also been demonstrated that the primers can be used in whole cell PCR for identification and whole cell PCR product recovery could be enhanced by the addition of gelatin or DMSO to PCR reaction mixtures. Other results have indicated that the method can be used for the definite identification of specific individuals present in mixed cultures or suspensions of organisms. The applicability of the method for detection of a specific strain within a group of closely related organisms has also been investigated and for that sequence/organism the results suggest that the proposed method is indeed very specific and discriminative. It is suggested that as more information becomes available regarding such sequences and their distribution, this approach could form the basis of a widescale, rapid, simple and cheap identification and/or typing system for bacteria.  1997 Academic Press Limited

KEYWORDS: bacteria, PCR, identification.

INTRODUCTION This report describes observations from studies on the use of a novel class of target sequences, which are readily identifiable and widely distributed amongst bacteria and which have so far been observed to demonstrate little similarity between different organisms,1 class II restriction endonucleases (RE), in PCR mediated microbial identification. Rapid, definitive, microbial identification and/or typing is desirable for a variety of industrial, medical, environmental, quality and research reasons. Traditionally identification has relied upon characteristics such as cell morphology and physiology and enzymic tests supplemented, more recently, with

information such as sensitivity to bacteriophages, cell wall composition/serotyping and DNA/RNA homologies and base ratio contents. (For examples of classical methodologies see Bergey (1994)2. Unfortunately, even when used in association such methodologies do not always guarantee positive identification of all bacterial types. Additionally, they are often time consuming, costly and technically complex to perform, requiring trained specialist personnel and highly equipped laboratory facilities. Some of the classical methodologies have now been semi-automated by API3 and Enterotube4 but with no greater guarantee of positive identification

∗ Author to whom correspondence should be addressed at: The Norwood Laboratories School of Chemical and Life Sciences, University of Greenwich, London, SE18 6PF, UK

0890–8508/97/040297+12 $25.00/0/mcpll970120

 1997 Academic Press Limited

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even though interpretation of results from such systems can be computer aided.5 Recently, there has been growth in rapid methods employed in microbial identification ranging from kits containing necessary reagents for the identification of specific bacterial genera or species to complicated and costly equipment. A wide variety of methodologies are represented in these products ranging from Direct Epifluorescence Microscopy6 of stained micro-organisms and enzyme linked immunoassays7 to DNA hybridization8 and bioluminescent technology, e.g. LUX.9 The polymerase chain reaction (PCR) has been and is being employed as a route to microbial identification and typing.10–15 So far three distinct methodologies have evolved: (1) DNA amplification fingerprinting16 (DAF), analogous to the method of Williams et al.17; (2) the use of ribosomal (rRNA) sequences and universal primers18; and (3) species or genus specific target sequences.19–22 However, each method has disadvantages such that method (1) can produce results which are difficult to interpret because of complex gel banding patterns with intensities that can be variable depending on template and primer concentrations. Method (2) generally requires hybridization of amplification products with an homologous probe for positive identification. Method (3) is limited by the total number of genus or species target sequences available at any time for use in identification. This report presents data that suggests that microbial identification could be based upon DNA sequences of class II restriction enzymes (RE). Class II RE’s are widely distributed amongst bacteria and so far 2393 have been identified from different prokaryote microbes23 which includes most major pathogens and micro-organisms of industrial and environmental relevance. Different REs have been identified among different bacterial families, genera and species and REs may even display differences at the level of bacterial serotypes.1,24–27 From current DNA sequence data available (see Table 1) it appears that most class II REs are greatly dissimilar, even isoschizomers, and that only relatively few show any significant DNA homology, e.g. RsrI and EcoRI.28 Only two REs have so far been observed to possess identical DNA sequences, Sso47I and EcoRI, the former possessed by Shigella sonnei strain 47 and the latter by E. coli KY13. Sso47I is known to be plasmid encoded but the strain also possesses another RE Sso47II which is unique to the organism. Although this is the only reported example of duplication of the same class II RE sequence in two different organisms, this particular strain possesses another characteristic restriction endonuclease by which it could be identified.

Many REs are commercially available and sequence data, both DNA and protein, already exists for some of them.29–31 So far approximately 90 have been sequenced as determined by accession information from EMBL and GenBank sequence databases and journal publications (Table 1). This has helped in the selection, construction and use of primers for this work. The organisms involved in this study were selected opportunistically with the exception of P. vulgaris possessing the RE PvuII. The latter was chosen for special attention to investigate the ability of the method to discriminate between closely related individuals within a defined group. Proteus and related organisms, Morganella and Providencia form a well defined, related group of organisms whose type and other strains are widely available from culture collections making for ease of study.

MATERIALS AND METHODS Bacterial strains See Table 2.

Cell culture, nucleic acid purification and PCR PCR amplifications of DNA sequences encoding the REs listed in Table 3 were carried out on whole cells, cell lysates and purified genomic DNA using the primers also described in Table 3. Primers were designed using OligoTM software32 and synthesized on an AB1 391 PCR-MATE DNA synthesizer. Cell lysates were prepared by sonication of a suspension of the desired organism of known cell density in 1 ml of sterile double distilled water at 4°C for 3 min in an MSE model L667 sonicator (MSE, London, UK). Cell debris subsequently removed by centrifugation and the supernatant used in DNA amplification. Total cellular DNAs were prepared by the method of Marmur.32 Cells employed in amplification reactions were recovered from 16 h cultures grown in the National Collection of Industrial and Marine Bacteria (NCIMB) recommended medium or LB broth at 37°C. Amplification reactions were carried out in 0·5 ml Eppendorf tubes and were organized as follows. Either x cells of the desired organism were suspended in 10 ll of sterile double distilled water or 10 ll of a cell lysate produced as described above or x ng of genomic DNA was added to oligonucleotide primers at a concentration of 0·2 l, 1 U of Taq DNA polymerase (Boehringer-Mannheim UK Ltd, Lewes, UK) with reaction buffer, each dNTP at 250 l and

Microbial detection using gene amplification Table 1.

299

Available RE DNA sequences

EMBL accession number

Enzyme

Organism including strain designation where provided

RE type

Location

D10671 X62690 X55285 D00704 X60713 L17341 L07643 X62104 S52585/L01541 X02988 M86639 X17591 X74517 Y00449 J01675 M74821 X05050 X00530/K02335 L18758 V00286/J01630 V00287/J01631 X73984 V00288/J01632 J03162/L18759 X13145 M27781/2 Z19104 M24927 X06287/X07312 X06288/X07313 J04623 D17388 K00508 M22862 X52124 D10668/D01166 X55139/S41812 X55138/S77155 X55142 X55143 X55137 X55140 M76435 X68366

AccI AbrI BamHI BanI BcreIII BcgI BstVi BsuFi BsuRI BsuRI CviAI CfrAI Cfr91 DdeI EcoRI Eco571 EcoRII EcoRV EcoAI EcoBI EcoD1 EcoDXXI EcoKI EcoEI EcoR124/31 Eco124I mrcA mcrB EcoPI EcoP15I FokI HgaI HhaII HinfI HincII HpaI HgiCII HgiCI HgiEI HgiGI HgiBI HgiDI KpnI PfvI

IIP IIW IIP IIP III IIS IIP IIP IIP IIP IIP IA (or IB) IIP IIW IIP IIS IIW IIP IA (or IB) IK (or IA) IK (or IA) R124 (or IC) IK (or IA) IA (or IB) R124 (or IC) R124 (or IC) II II III III IIS IIS IIW IIW IIP IIP IIW IIP IIW IIP IIW IIW IIP II?

U U U C C U C C C U V C U U P U P P C C C P C C P P C C P P C U U U C U C U U U U U U P

D13968 X56977 X14191 X76192 L14564 X52661 D14719 U03474 L04163 X52681 K02081 X03274 X14697 J03391

MboI MboII MspI MunI dcrB NgoPII NspV PvuRtsI PvuI PvuII PstI PaeR71 RsrI SinI

Acinetobacter calcoaceticus Azospirillum brasilense Bacillus amyloliquefaciens H Bacillus aneurolyticus IAM 1077 Bacillus cereus ATCC 10987 Bacillus coagulans Bacillus stearothermophilus V Bacillus subtilis IAM 1192, 1231 Bacillus subtilis IAM 1247 Bacillus subtilis R IAM 1076, 1114 Chlorella strain NC64A virus PBCV-1 Citrobacter freundii NCTC 9750 Citrobacter freundii RFL9 Desulfovibrio desulfuricans Norway Escherichia coli RY13 (plasmid pMB?) Escherichia coli RFL57 Escherichia coli R245 (plasmid N3) Escherichia coli J62 (plasmid pLB1) Escherichia coli 15T Escherichia coli B Escherichiacoli E166 Escherichia coli HB101 (plasmid DXXI) Escherichia coli K Escherichia coli A58 Escherichia coli (plasmid R124/3) Escherichia coli (plasmid R124) Escherichia coli K12 Escherichia coli K12 Escherichia coli (plasmid PI) Escherichia coli (plasmid P15B) Flavobacterium okeanoites IFO 12536 Haemophilus gallinarum NCTC 3438 Haemophilus haemolytics ATCC 10014 Haemophilus influenzae Rf Haemophilus influenzae Rc Haemophilus parainfluenzae Herpetosiphon giganteus Hpg9 Herpetosiphon giganteus Hpg9 Herpetosiphon giganteus Hpg24 Herpetosiphon giganteus Hpa1 Herpetosiphon giganteus Hpg5 Herpetosiphon giganteus Hpa1 Klebsiella pneumoniae OK8 Methanobacterium thermoformicum THF (plasmid pFV1) Moraxella bovis ATCC 10900 Moraxella bovis ATCC 10900 Moraxella spp. Mycoplasma spp. Neisseria gonorrhoeae F62 Neisseria gonorrhoeae P9-2 Nostoc spp. PCC 7524 Proteus vulgaris UR75 (plasmid Rts1) Proteus vulgaris ATCC 13315 Proteus vulgaris ATCC 13315 Providencia stuartii 164 Pseudomonas aeruginosa PA0303 (plasmid pMG7) Rhodobacter sphaeroides RS630 Salmonella infantis

IIP IIS IIP II? IIP IIP IIP II? IIP IIP IIP IIP IIP IIW

C C U U C C U P U U U P U U continued

L. A. Metherell et al.

300 Table 1.

Available RE DNA sequences—continued

EMBL accession number

Enzyme

Organism including strain designation where provided

RE type

Location

M14984 M15940

StySPI StySQI

IK (or IB) IK (or IB)

C C

Y00524 M90544 X16458 M97479 X53096 M32470 M14340 M14339 D11101 U01232 M74796 M74795 M98768 Ref. 36 Ref. 37 Ref. 38

StySBI StyLTI SmaI Sso47I Sau961 Sau3AI Dpn1 DpnII StsI SalI TaqI TthHB81 XcyI CfrBI HpaII Sso47II

Salmonella potsdam L4002 Salmonella potsdam/typhimurium recombinant L4004 Salmonella typhimurium L4001 Salmonella typhimurium LT7 Serratia marcescens S-b Shigella sonnei 47 (plasmid P6) Staphylococcus aureus PS96 Staphylococcus aureus 3A Streptacoccus pneumoniae (formerly Diplococcus) Streptococcus pneumoniae Streptococcus sanguis 54 Streptomyces albus G Thermus aquaticus YT1 Thermus thermophilus HB8 Xanthomonas campestris 13D5 Citrobacter freundii Haemophilus parainfluenzae Shigella sonnei 47 (plasmid P4)

IK (or IB) III IIP IIP IIW IIP IIP IIP II? IIP IIP IIP IIP II? IIP IIW

C C U P U U U U C U C U U U U P

U=undetermined; P=plasmid; V=virus; C=chromosomal; ?=uncertain. Data was abstracted from EMBL and GenBank sequences databases and other published works.

sterile double distilled water to make up to a final volume of 100 ll. All reactions except those for PvuII primers were cycled without oil overlay in a Techne PHC3 thermal cycler with heated lid (Techne (Cambridge) Ltd, Duxford, UK) and results for individual PCR reactions were generated using three temperature cycling. This involved denaturation for 2 min at 95°C, primer annealing at 65°C for 30 s, and extension for 50 s at 72°C followed by 30 cycles consisting of a 30 s denaturation step at 95°C, a primer annealing step of 30 s at 65°C and a 60 s primer extension step at 72°C. The last cycle consisted of a 30 s denaturation step at 95°C, a 30 s primer annealing step at 65°C and a 5 min extension step at 72°C. Multiplex PCR was carried out by ‘Touchdown’ PCR in which the annealing temperature was reduced from 70–58°C over the first 12 cycles by 1°C per cycle. Otherwise conditions for multiplex PCR were identical to those described previously. Post reaction, 20 ll of the mixture was removed and analysed by electrophoresis with loading dye in a 1·5% w/v agarose slab gel. Reactions using PvuII primers involved an altered protocol which consisted of an initial denaturation for 5 min at 95°C followed by 30 cycles of 95°C for 30 s, 63°C for 30 s and 72°C for 60 s. Finally, the reaction was held at 72°C for 5 min. A 10 ng aliquot of template DNA was used in these reactions.

RESULTS To test the sensitivity and definition of microbial identification using RE primers we designed (using OligoTM software32) and synthesized forward and reverse primers to the RE DNA sequences listed in Table 3. Amongst these were EcoRI and RsrI whose DNA sequences are known to possess high homology28 and represent the most homologous REs observed to date (with the exception of EcoRI and Sso47I which have been reported to be identical) and as such, should have been a suitably stringent test of the proposed system. All RE sequences were compared against each other and the total EMBL and GenBank databases for sequence similarity, as were primers designed for use in amplifications. No significant sequence homologies were observed between any primers and REs for which DNA sequence data is available other than with their target sites. Table 3 lists the primers used in experiments and the sizes of amplification products generated by their use. The primers have all been successfully used individually in the amplification of 680, 326, 109, 727, 264, 611 and 488 bp fragments of the genes encoding EcoRI, RsrI, BamHI, HpaI, KpnI, Sau3A and SinI and together in multiplex PCR (Fig. 1). These experiments have been carried out on purified genomic DNA,

Microbial detection using gene amplification Table 2.

301

Bacterial strains

Organism

Strain/Serovar

NCIMB

Bacillus cereus Bacillus laterosporus Bacillus megaterium Bacillus stearothermophilus Bacillus subtilis Bacillus subtilis Bacteroides fragilis Chromobacterium violaceum Citrobacter freundii Clostridium butyricum Clostridium difficile Clostridium perfringens

Lederle 5 Ford 29 Ford 19

8122 9367 9376 8922 3610 10113

Clostridium sporogenes Clostridium thermosaccharolyticum Desulfotomaculum nigrificans Enterobacter aerogenes Enterococcus faecalis Erwinia R Escherichia Escherichia Escherichia Escherichia Escherichia

coli coli coli coli coli

Hafnia alvei Klebsiella pneumoniae Klebsiella pneumoniae Lactobacillus brevis Lactobacillus plantarum Lactococcus lactis Legionella anisa Legionella bozemanii Legionella cherrii Legionella gormanii Legionella israelensis Legionella longbeachae Legionella longbeachae Legionella parisiensis Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella steigerwaltii Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria seeligeri Micrococcus luteus Micrococcus luteus Micrococcus roseus

Marburg 166 type Rettger 4/22 Braak Rowett type Cambridge SR12 McClung 2003 McClung 2302 Teddington Garden A-29 P-60

NCTC

Other sources

9343 8890 3735 7423 11209 6784 8053 9385 8351 366 7432 Dr A. W. Smith Univ of Greenwich London, England

type C3 Escherich Roepke 1572-228 RY13

9107 8277 86 8876 Dr R. Roberts New England Biolabs MA, USA 8535

240 C37 118-8 lactis

418 8267 947 8960 8586 11974 11368 11976 11401 12010 11477 11530 11983 11404 11232 11231 11286 11287 11192 11191 11230 11991 4883 4885 4886 7973 10357 11856

Bellingham Bloomington Cambridge Knoxville Oxford Philadelphia Pontaic Togus-1

type type Stanley 130.21 213

8553 8166 8175 continued

L. A. Metherell et al.

302 Table 2.

Bacterial strains—continued

Organism

Strain/Serovar

Morganella morganii Pasteurella multocida Proteus penneri Proteus mirabilis Proteus myxofaciens Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Proteus vulgaris Providencia rettgeri Providencia stuartii Providencia haimbachae Providencia alcalifaciens Providencia fredericiana Providencia rustigianii Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas spp. Rhodobacter sphaeroides Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella

enterica enterica enterica enterica enterica enterica enterica enterica enterica enterica enterica enterica enterica infantis

Salmonella typhimurium Serratia marcescens Serratia marcescens Shigella boydii Shigella dysenteriae Shigella flexneri Shigella flexneri Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus 3A Staphylococcus aureus Staphylococcus epidermidis Streptococcus pyogenes Streptococcus pyogenes

NCIMB

NCTC

Other sources

235 10322

type X19

12597 60 13273 4175 67 8066 8065 8259 13111 8064 11833 1334 7052 8067 8261 9570 11800 12003 10286 11667 11802 10322

type Spiers

8295 3756 9340

RS630

Dr Roberts New England Biolabs MA, USA

abortusequi berta dahlem derby lexington london montevideo paratyphi poona stanley sundsvall thompson weslaco

5727 5770 9949 5721 6244 5777 5747 3176 4840 92 6758 5740 7411 Dr A. de Waard Oegstgeest, Netherlands 5710 9155

type

10211 9333 9955 8517 9729 6507 7856 8532

type 8625

Dr R. Roberts New England Biolabs MA, USA 3R7089 type C203M

6571 8853 8198 8884

HpaI EcoRI Sau3A SinI RsrI KpnI BamHI PvuII

1 Haemophilus parainfluenzae

2 Escherichia coli RY13

3 Staphylococcus aureus 3A

4 Salmonella infantis

5 Rhodobacter sphaeroides RS630

6 Klebsiella pneumoniae OK8

7 Bacillus amyloliquefaciens H

8 Proteus vulgaris NCIMB 4175

Restriction enzyme

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse AGT CCA GCT AAC CAG TTT CCC TGT GAC GCA TTC CAG AAA CAT ACA ACC

Primer sequences used in amplifications of restriction enzyme sequences

Organism

Table 3.

GCC TTT CTT AAG ACA CTT ACT GAT CAA AGA CCA CGT AGG TGA GCC AGG

TGT ATT AGC TCA TAA CAC CAT GTC GAA AGA GGT ATT TGG ACG CTC ACG

AGA TCT AAC CGC AAG TGT CTT AGA GGC CGA TTC GAA TCC GTG AAA AGA

GTC TTT GAA CCC CAA ATC TTT GGC AAT CAT TTT GCC GAT GGC CGT AGG

CCC TGC TAC AAC AGC ATC CAT AGT CAA AGG TGT CTA TGA AGA CAC TAA

TTA CTC CCT ACT AAA CCA TGT GTT CGA GGA CGG TTT TGT ACT AGA TGA

Primer sequence

TTA ATT CAA CTG GAG CAT CAT TTT AAA AAT TAA CTT TTA AAT AA TG

C A T A C T A C G G T A T A

505

109

264

326

488

611

680

727

Amplification fragment size (bp)

Microbial detection using gene amplification 303

L. A. Metherell et al.

304 1

2

3

4

5

6

7

8

9

10

1000 bp 900 800 700 600 500 400 300 200 100

Fig. 1. Single and multiplex PCR amplification of RE DNA sequences in whole cells, cell lysates and genomic DNA. PCR amplification of DNA sequences encoding the REs listed in Table 3 were carried out on whole cells, cell lysates and purified genomic DNA using the primers also described in Table 3. Amplification reactions and agarose gel electrophoresis were performed as described in the Materials and Methods section. Lane 1, 100 bp ladder (size marker); 2, HpaI primers and Haemophilus parainfluenzae; 3, EcoRI primers and Escherichia coli RY13; 4, Sau3A primers and Staphylococcus aureus 3A; 5, SinI primers and Salmonella infantis; 6, RsrI primers and Rhodobacter sphaeroides RS630; 7, KpnI primers and Klebsiella pneumoniae OK8; 8, BamHI primers and Bacillus amyloliquefaciens H; 9, multiplex of all primers and template DNAs; 10, 100 bp ladder (size marker).

Table 4.

Results from sensitivity experiments involving amplification of purified DNA and whole cells using RE primers

RE primers

Organism

BamHI EcoRI HpaI KpnI RsrI Sau3A SinI

Bacillus amyloliquefaciens H Escherichia coli RY13 Haemophilus parainfluenzae Klebsiella pneumoniae OK8 Rhodobacter sphaeroides RS630 Staphylococcus aureus 3A Salmonella infantis

DNA concentration (g)

Cell number

10−18 10−18 10−9 10−18 10−18 10−12 10−12

1[vΖ10 1[vΖ10 100[vΖ1000 10[vΖ100 1[vΖ10 100[vΖ1000 1[vΖ10

DNAs were purified and cell numbers estimated by the methods described previously. 10-fold serial dilutions of cells and DNA in PCR reaction buffer were constructed between 10−7 and 10−20 g DNA and 1 to 106 cells (see Materials and Methods). Amplifications were performed as previously described and product detection was by agarose gel analysis. DNA concentration represents the minimum amount required to produce visible bands after amplification and gel electrophoresis and v is the range within which the minimum number of cells exist to achieve a similar observation. v is an average value from five experiments.

Microbial detection using gene amplification 1

2

3

4

5

6

7

8

9

305 10

11

12

13

14

1000 bp 900 800 700 600 500 400 300 200 100

Fig. 2. Amplification of PvuII RE DNA sequence. PCR amplification of DNA (10 ng) from selected Proteus species was carried out using PvuII primers as described in the Materials and Methods, the negative control lacked template DNA. Products were analysed by agarose gel electrophoresis as previously described. Lane 1, 100 bp ladder (size marker); 2, Proteus vulgaris NCIMB 67; 3, Proteus vulgaris NCIMB 8066; 4, Proteus vulgaris NCIMB 8065; 5, Proteus vulgaris NCIMB 8259; 6, Proteus vulgaris NCIMB 13111; 7, Proteus vulgaris NCIMB 8064; 8, Proteus vulgaris NCIMB 11833; 9, Proteus vulgaris NCIMB 1334; 10, Proteus vulgaris NCIMB 7052; 11, Proteus vulgaris NCIMB 8067; 12, Proteus vulgaris NCIMB 8261; 13, Proteus vulgaris NCIMB 4175 type strain; 14, negative control.

crude cell lysates and whole cells and similar results have been obtained from all three. Figure 1 is a typical observation from such experiments. Table 4 illustrates the results of sensitivity studies performed using the primers in PCR on purified genomic DNAs and whole cells. Cell numbers refer to values from total cell counts made using a haemocytometer slide and phase contrast microscope. It was observed that PCR product concentration from reactions involving whole cells could be increased by the addition of either gelatin or DMSO to amplification reactions at concentrations of 0·5 mg ml−1 and 15% v/v, respectively. Between 1 and 1000 cells of the organisms used, depending on the strain and primer combination, and attogram amounts of purified genomic template DNA were detectable. Using this group of eight organisms, expriments have also been conducted to investigate the possibility of cross-annealing of the primer pairs to non-target DNAs. Such experiments involved using each individual primer pair in amplifications of all non-target DNAs. No PCR product and therefore cross-annealing of the primers to non-target DNAs has been observed under the conditions for PCR given in Materials and Methods. In the case of EcoRI and Rsr I it is known that the REs are encoded by gene sequences that show 50% identity within a 266 amino acid overlap and up to 75–100% sequence identity in regions

corresponding to key enzyme structural and functional areas28 and it is therefore reasonably safe to conclude that with optimized primer design even closely related REs and, as a result, their host organisms can be discriminated. Touchdown PCR was employed in multiplex reactions as it was observed to increase both specificity of primer annealing and product concentration. The RE primers used in these experiments have also been tested against a wide range of closely and distantly related micro-organisms in PCR reactions (see Table 2) and no positive results using the protocols outlined in the Materials and Methods, i.e. amplification products, have been obtained in any such experiment employing either whole cells or cell lysates. The organisms listed in Table 2 represent the range available in, and to, our laboratory. To test the efficiency of the method in discriminating closely related organisms, primers designed to amplify a 505 bp region of the PvuII gene from P. vulgaris NCIMB 4175 (the type strain and host for the PvuII RE) were used in amplification reactions of DNA purified from that organism and all other P. vulgaris strains commercially available. The results are illustrated in Fig. 2. Only P. vulgaris NCIMB 4175 yielded the expected results. The amplification fragment was confirmed as the expected product by its digestion with XbaI, which is known to cut the

L. A. Metherell et al.

306 1

2

3

4

5

6

7

8

9

10

11

12

13

1000 bp 900 800 700 600 500 400 300 200 100

Fig. 3. Amplification of PvuII RE DNA sequence. PCR amplification of DNA (10 ng) from selected genera and species closely related to Proteus vulgaris was carried out, as referred to in Fig. 2. Lane 1, 100 bp ladder (size marker); 2, Morganella morganii NCIMB 235; 3, Proteus penneri NCIMB 12597; 4, Proteus mirabilis NCIMB 60; 5, Proteus myxofaciens NCIMB 13273; 6, Providencia rettgeri NCIMB 9570; 7, Providencia stuartii NCTC 11800; 8, Providencia haimbachae NCTC 12003; 9, Providencia alcalifaciens NCTC 10286; 10, Providencia fredericiana NCTC 116677; 11, Providencia rustigianii NCTC 11802; 12, Proteus vulgaris NCIMB 4175 type strain; 13, negative control.

sequence internally at a single site to generate products of 296 and 209 bo (data not shown). Proteus vulgaris is a member of the family Enterobacteriaceae and the genus Proteus is particularly closely related to the genera Morganella and Providencia.2 It was decided to test the primers in amplification reactions using purified DNAs from the type strain and all strains of each of the genera noted above that were available and other Proteus spp. (see Figs 2 and 3). In amplifications carried out under the conditions described for experiments involving PvuII primers noted in the Materials and Methods none of the other organism’s DNAs tested were found to produce amplification products. When the annealing temperature was reduced to 55 or 50°C many bands were observed after agarose gel electrophoresis of amplification products, but none possessed the expected size. However these bands were removed from the agarose gels and subjected to XbaI digestion, none produced the expected digestion product sizes.

DISCUSSION The results reported here suggest that RE DNA sequences can be used for the specific detection of their host organisms and that by using these sequences the levels of detection compare favourably with those published by others for PCR sensitivity.19,34,35 The results from whole cell studies may also suggest that either the bacterial cell wall is permeable to

oligonucleotide primers or that cell lysis may have occurred during thermal cycling allowing access of primers to target DNA. The results from studies involving primers designed to amplify a PvuII gene fragment suggest that the method is highly discriminatory even when applied to closely related organisms. The implication of these results is that the PvuII gene is not widely distributed within this genus or other closely related genera. This specificity could limit the methods applicability as a general way of bacterial identification. It is known that some REs are distributed on plasmids and so far no comprehensive studies have been carried out to determine the breadth of their potential host range or if a particular type of plasmid is involved. For example, it is known that EcoRI is plasmid encoded and that S. sonnei also possesses the same enzyme (Sso47I). This particular situation may have arisen as Shigella and Escherichia can be closely related and are able to exchange genetic material. In the case of the S. sonnei strain host to Sso471 the organism is known to possess a second plasmidencoded RE which so far has been identified as strain specific. However, it may be that distribution of RE plasmid encoded genes is limited. For example, the exact location of the gene encoding PvuII in P. vulgaris has yet to be established, but if it is proven to be plasmid-borne then the results reported here may suggest that its host range is very limited. In summary, class II REs can be diagnostic for bacteria, although more work is needed to make

Microbial detection using gene amplification

this a general method for microbial identification. At present, the method is a useful addition to the other approaches available for microbial detection.

ACKNOWLEDGEMENTS We wish to thank the University of Greenwich for support of LAM during the course of this work, Techne (Cambridge) Limited for the supply of a PHC3 thermal cycler and Miss J. Crow for typing the manuscript. We are also grateful to Richard Roberts and Adrian de Waard for supplying some of the strains used in this work and for helpful comments.

REFERENCES 1. Wilson, G. G. & Murray, N. E. (1991). Restriction and modification system. Annual Review of Genetics 25, 585–627. 2. Bergey’s Manual of Determinative Bacteriology (1994). Williams and Wilkins Company: USA. 3. A.P.I. Systems S.A., La Balme Les Grottes, 3890 Montalieu Vercill, France. 4. Enterotube, F. Hoffman La Roche and Cie S.A., Diagnostica, Basle, Switzerland. 5. APILAB Plus, A.P.I. Systems S.A., La Balme Les Grottes, 3890 Montalieu Vercill, France. 6. Pettipher, G. L. (1983). The Direct Epifluorescent Technique for Rapid Enumeration of Micro-organisms. Letchworth Research Studies Press. 7. Bio-Enzabead Screen Kit, Organon Teknika Ltd., Cambridge, MA, USA. 8. Gen-Probe Inc., 9880 Campus Point Drive, San Diego, CA, USA. 9. Engebrecht, J. M. S. & Silverman, M. (1985). Measuring gene expression with light. Science 227, 1345–7. 10. Maiwald, M. & Zoller, L. (1991). The polymerase chain reaction in the bacteriological diagnostic laboratory—an enlarging spectrum of applications. Klinisches Labor 37, 194–200. 11. Atlas, R. M. (1991). Environmental applications of the polymerase chain reaction. ASM News, 630–2. 12. Steffan, R. J. & Atlas, R. M. (1991). Polymerase chain reaction: applications in environmental microbiology. Annual Review of Microbiology 45, 137–61. 13. Atlas, R. M., Sayler, G., Burlage, R. S. & Bej, A. K. (1992). Molecular approaches for environmental monitoring of micro-organisms. Biotechniques 12, 706–16. 14. Bruce, I. J. (1994). Nucleic acid amplification mediated microbial identification. Science Progress 77, 183–206. 15. Hill, W. E. (1996). The polymerase chain reaction: applications for the detection of foodborne pathogens. Critical Reviews in Food Science and Nutrition 36, 123–73. 16. Welsh, J. & McClelland, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213–8. 17. Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. & Tingey, S. V. (1991). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531–5.

307

18. Wilson, K. H., Blitchington, R. B. & Green, R. C. (1990). Amplification of 16S ribosomal DNA with polymerase chain reaction. Journal of Clinical Microbiology 28, 1942–6. 19. Bej, A. K., Steffan, R. J., DiCesare, J., Haff, L. & Atlas, R. M. (1990). Detection of Coliform bacteria in water by polymerase chain reaction and gene probes. Applied and Environmental Microbiology 56, 307–14. 20. Houard, S., Hackel, C., Herzog, A. & Bollen, A. (1989). A Specific Identification of Bordattella pertussis by the polymerase chain reaction. Research in Microbiology 140, 477–87. 21. Mahubani, M. H., Bej, A. K., Miller, R., Haff, L., DiCesare, J. & Atlas, R. M. (1990). Detection of Legionella with polymerase chain reaction and gene probe methods. Molecular and Cellular Probes 4, 175–8. 22. Moter, S. E., Kramer, M. D., Simon, M. M., Schaible, U. E., Kinzelbach, S. R. & Wallich, R. (1991). PCR Topics. (Rolfs, A., Schumacher, H. & Marx, P.). Pp 206–213. Berlin: Springer-Verlag. 23. Roberts, R. J. & Macelis, D. (1993). REBASE— restriction enzymes and methylases. Nucleic Acids Research 21, 3125–37. 24. Roberts, R. J. (1976). Restriction enzymes. Critical Reviews in Biochemistry 4, 123–64. 25. Kessler, C. & Manta, V. (1990). Specificity of restriction endonucleases and DNA modification methyl transferases—a review (edition 3). Gene 92, 1–248. 26. Roberts, R. J. (1990). Restriction enzymes and their isoschizomers. Nucleic Acids Research 18, r271–r316. 27. Szybalski, W., Kim, S. C., Hasan, N. & Podhajska, A. J. (1991). Class IIs restriction enzymes. Gene 100, 13–26. 28. Stephenson, F. H., Ballard, B. T., Bayer, H. W., Rosenberg, J. M. & Greene, P. J. (1989). Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases. Gene 85, 1–13. 29. Looney, M. C., Moran, L. S., Jack, W. E. et al. (1989). Nucleotide sequence of the FokI restriction modification system: separate strand specificity domains in methyl transferase. Gene 80, 193–208. 30. Szilak, L. & Kiss, A. (1990). Cloning and nucleotide sequence of the genes coding for the Sau961 restriction and modification enzymes. Nucleic Acids Research 18, 4659–64. 31. Maekawa, Y., Yasukawa, H. & Kawakami, B. (1990). Cloning and nucleotide sequences of the BanI restriction modification genes in Baccilus aneurinolyticus. Journal of Biochemistry 107, 645–9. 32. OligoTM, National Biosciences Inc., Plymouth, MN, USA. 33. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. Journal of Molecular Biology 3, 208–18. 34. Olive, D. M., Atta, A. I. & Setti, S. K. (1988). Detection of toxigenic E. coli using biotin labelled DNA probes following enzymatic amplification of the heat labile toxin gene. Molecular and Cellular Probes 2, 47–57. 35. Pillai, S. D., Josephson, K. L., Bailey, R. L., Gerba, C. P. & Pepper, I. L. (1991). Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Applied and Environmental Microbiology 57, 2283–6. 36. Zakharova, M. V., Kravetz, A. N., Beletzkaja, I. V., Repyk, A. V. & Solonin, A. S. (1993). Cloning and

308

L. A. Metherell et al.

sequences of the genes encoding CfrBI restriction modification system from Citrobacter freundii. Gene 129, 77–81. 37. Kulakausakas, S., Barsomian, J. M., Lubys, A., Roberts, R. J. & Wilson, G. G. (1994). Organisation and sequence of the HpaII restriction modification system and adjacent genes. Gene 142, 9–15.

38. Karyagina, A. S., Lunin, V. G., Degtyarenko, K. N., Uvarov, V. Y. & Nikolskaya, I. I. (1993). Analysis of the nucleotide and derived amino acid sequences of the SSoII restriction endonuclease and methyl transferase. Gene 124, 13–19.