JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1994, p. 3002-30070095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Rapid Identification of Bacteria by PCR-Single-StrandConformation Polymorphism

MYRA N. WIDJOJOATMODJO, 12* AD C. FLUIT,"12 AND JAN VERHOEF'

Eijkman Winkler Institute for Medical and Clinical Microbiology, University Hospital Utrecht,'and U-gene Research B. V,2 Utrecht, The Netherlands

Received 9 May 1994/Returned for modification 14 July 1994/Accepted 15 September 1994

A new molecular biological approach for the identification of bacteria is described. This approach employsPCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variableregion, and analysis of the amplified produ-ct was based on the principle of single-strand conformationpolymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamidegel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNAis sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was

demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains ofbacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P andER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specificpatterns were obtained for, e.g., Clostridium spp., Listeyia spp., Pseudomonas spp., and Enterobacter spp.

PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. Thistechnique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria bywhole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gelelectrophoresis.

Nucleic acid-based amplification systems like PCR arepromising methods for the rapid detection of low numbers ofpathogenic bacteria. The 16S rRNA sequence, which is highlyconserved throughout the phylogenetic tree (12), is found in allprokaryotic organisms and is one of the most extensivelystudied target sequences. The 16S rRNA gene also containsvariable regions which have been used for discriminationbetween species and genera. The conserved sequences of the16S rRNA have led to the development of conserved primersfor PCR for the detection of eubacteria (7, 25). PCR amplifi-cation with conserved primers followed by sequencing of thevariable regions has enabled the development of specificprobes (2). Recently, Greisen et al. (11) proposed the use ofPCR with conserved primers followed by hybridization with aseries of probes for the detection of bacteria in cerebrospinalfluid. The first series of probes consisted of a specific probe forgram-positive bacteria and a specific probe for gram-negativebacteria and was followed by a second round of hybridizationwith species- or genus-specific probes. However, the majordisadvantage of this method is the need for a comprehensivepanel of probes or prior knowledge of the expected microor-ganism. Other novel DNA-based identification methods forbacteria include denaturing gradient gel electrophoresis anal-ysis of PCR-amplified regions (18) and amplification of thespacer regions between the 16S and 23S rRNA genes (14);both systems result in species-specific electrophoresis patterns.

Here, we present a new, general approach for directlydetermining the identity of bacteria based on the principle ofsingle-strand conformation polymorphism (SSCP) electro-phoresis of PCR-amplified 16S rRNA fragments. SSCP is a

technique designed to detect mutations in oncogenes and

* Corresponding author. Mailing address: Eijkman Winkler Insti-tute for Medical and Clinical Microbiology, University Hospital Utre-cht, Room G04.614, P.O. Box 85500, 3508 GA Utrecht, The Nether-lands.

allelic variants in the human genome (19, 20). After PCRamplification of the target sequence, the amplified product isdenatured to two single stranded DNAs (ssDNAs) and sub-jected to nondenaturing polyacrylamide gel electrophoresis.Under nondenaturing conditions, ssDNA has a secondarystructure that is determined by the nucleotide sequence. Themobility of the ssDNA depends on the secondary structure ofthe amplified product. Bands of ssDNA at different positionson the gel indicate a different sequence. PCR-SSCP is capableof detecting >90% of all single-base substitutions in 200-bpfragments (13). We tested the conserved 16S rRNA primersusing PCR of cell lysates from more than 100 bacterial strainsand subjected them to SSCP. The obtained electrophoreticpatterns were then analyzed.

MATERIALS AND METHODS

Bacterial strains. Bacterial strains used were either clinicalisolates collected from the University Hospital Utrecht (Utre-cht, The Netherlands) or the National Institute for PublicHealth and Environmental Protection (RIVM, Bilthoven, TheNetherlands) or isolates obtained from the American TypeCulture Collection. Clinical isolates were identified by Gramstaining and biochemical tests using standard assays.

Bacterial strains were cultured overnight at 37°C on bloodagar plates, scraped from the plates, and lysed in a mixture of10% Chelex 100 (Bio-Rad, Richmond, Calif.), 0.03% sodiumdodecyl sulfate, 1% Tween 20, and 1% Nonidet P-40 for 5 minat 95°C. After centrifugation for 10 s, 5 ,ul of the supernatantwas directly used for PCR amplification.The number of CFU was determined by plating on blood

agar and culturing for 16 h at 37°C.Primers. Oligonucleotide primers were synthesized on a

Pharmacia LKB Gene Assembler Plus synthesizer (PharmaciaAB, Uppsala, Sweden). The target DNA sequence was the 16SrRNA gene. The first set of primers was P13P (5'-AGG CCCGGG AAC GTA TTC AC) and P11P (5'-GAG GAA GGT

3002

Vol. 32, No. 12

on May 22, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

PCR-SSCP FOR BACTERIAL IDENTIFICATION 3003

without DNA template

A DNase

with DNA template

B DNase (PH)

M 1 2 3 4 5 6 7M

W J

- PH BM M - PH BM

FIG. 2. Sensitivity of PCR-SSCP with primer set P11P-P13P. Aserial dilution of E. coli cells ranging from 0 to 1,000 CFU was testedwith a 35-cycle PCR. Lanes 1 to 7, 0, 3, 10, 30, 100, 300, and 1,000 CFUof E. coli cells, respectively; lanes M, dsDNA molecular size markers(1,078, 872, and 603 bp).

C DNase (BM) 1 2 3 4 5 M 1 2 3 4 5

FIG. 1. Effect of DNase I treatment on the amplification reaction.Left lanes, Ampli-Taq polymerase; lanes M, marker lanes (lambda-PstIdigest); right lanes, Super-Taq polymerase. (A) Reactions withoutadded DNA template. -, no DNase treatment; PH, DNase I (Phar-macia); BM, DNase I (Boehringer Mannheim). (B and C) E. coli celllysates were added to the amplification reaction after DNase Iinactivation. Lanes 1 to 5, 30, 100, 300, 103, and 104 CFU of E. coli,respectively.

GGG GAT GAC GT) to amplify a 216-bp fragment of the V6region (Eschenichia coli 16S rRNA positions 1175 to 1390) (7).The second set of conserved primers was ER10 (5'-GGC GGACGG GTG AGT AA) and ER11 (5'-ACT GCT GCC TCCCGT AG) to amplify a 255-bp region of the V2 region (E. coli16S rRNA positions 104 to 358). After deprotection andcleavage, the oligonucleotides were purified by ethanol precip-itation.DNA amplification. The PCR mixture (50 ,ul) contained 50

mM Tris-HCl (pH 9.0), 50 mM KCl, 7 mM MgCl2, 2 mg ofbovine serum albumin per ml, 16 mM (NH4)2SO4, 100 ,uM(each) primer, and 0.1 U of Super-Taq polymerase (HTBiotechnology, Cambridge, England). The PCR mix was incu-bated with 0.5 ,ug of DNase I, which is active on double-stranded DNA (dsDNA; Boehringer, Mannheim, Germany),for 15 min at room temperature and then was subjected toDNase inactivation for 10 min at 95°C. Then 5 plI of the celllysate, containing target DNA, was added to the PCR mix, andPCR was performed for 30 cycles of 1 min at 94°C, 10 s at 72°C,and 1 min at 55°C on a DNA thermocycler (Perkin-ElmerCetus, Norwalk, Conn.). After amplification, 5 pl of theamplified product was run on a 1% agarose gel in 0.5 x TBE.DNA bands were detected by ethidium bromide staining andvisualized by UV light photography.

Other thermostable DNA polymerases included in this studywere Ampli-Taq polymerase (Perkin-Elmer Cetus), Native-Taq polymerase (Perkin-Elmer Cetus), Vent polymerase (NewEngland Biolabs, Beverly, Mass.), and Tth polymerase (U.S.

Biochemical Corp., Cleveland, Ohio). These thermostableenzymes were used with the buffer supplied by the producer.Another DNase I tested was from Pharmacia, which is activeon both ssDNA and dsDNA; elimination of exogenous DNAwas similar to Boehringer's DNase treatment, but primerswere added just after heat inactivation of the enzyme.The sensitivity of PCR-SSCP was determined by performing

the PCR for 35 cycles with cell lysates of E. coli and Staphylo-coccus aureus.SSCP electrophoresis. After thermal cycling, 1 to 10 RI of

the PCR mixture was added to 3 ,u of sequencing samplebuffer (5 mM EDTA, 0.05% bromophenol blue, 0.05% xylenecyanole in formamide) and heated for 5 min at 95°C. Thedenatured DNA was then placed directly onto ice for 10 minbefore being loaded onto the gel. The optimal gel compositionwas 0.5x mutation detection enhancement (MDE) gel (J. T.Baker, Phillipsburg, N.J.), 10% glycerol, 0.6x TBE, 0.05%APS, and 0.005% N,N,N',N'-tetramethylethylenediamine.Electrophoresis was performed at room temperature on eithera Bio-Rad Protean II or Mini-Protean electrophoresis appara-tus. Electrophoresis was performed on the Protean II over-night at 5-W constant power. For minigels, electrophoresis wascarried out for 3 h at 3-W constant power.

Detection of SSCP patterns. After electrophoresis, SSCPwas detected by silver staining according to the method ofBassam et al. (3). Briefly, the silver staining procedure was asfollows: the gels were first fixed in 10% acetic acid for 20 minat room temperature and washed with deionized water threetimes for 2 min each time. Color impregnation lasted for 30min at room temperature with 0.1% silver nitrate and 0.056%formaldehyde. The gels were then washed for 20 s withdeionized water; then color development was for 2 to 10 minwith a mixture of 30 g of sodium carbonate per liter, 0.056%formaldehyde, and 2 mg of sodium thiosulfate per liter. Thecolor reaction was stopped with 10% acetic acid.

RESULTS

Pre-PCR sterilization by DNase I. Contamination of PCRreagents, in particular Taq polymerase, with exogenous bacte-rial DNA is a major problem for the amplification of targetsequences like the 16S rRNA sequence with conserved primers(21). Therefore, we examined the use of pre-PCR sterilizationof the entire PCR mix without the target DNA by a short

VOL. 32, 1994

on May 22, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

3004 WIDJOJOATMODJO ET AL.

DNase I incubation. This strategy, however, requires properinactivation of DNase I prior to addition of the target DNA. Anumber of thermostable DNA polymerases were tested: Am-pli-Taq, Native-Taq, Super-Taq, Vent, and Tth polymerases, incombination with two different DNases I from different sup-pliers (Boehringer and Pharmacia). The best results withregard to the elimination of exogenous bacterial DNA andproper inactivation of DNase I were obtained only withBoehringer DNase I in Super-Taq polymerase PCR mixture(Fig. 1). Elimination of exogenous DNA with DNase I fromPharmacia hampered PCR amplification. Boehringer DNase Iin Ampli-Taq polymerase PCR mixture was able to eliminateexogenous DNA, but the PCR after DNase I treatment wasless sensitive than that with Super-Taq polymerase. DNase Iwas either incapable of eliminating exogenous DNA or notproperly inactivated when incubated with the other DNApolymerase mixtures tested (data not shown). Other inactiva-tion procedures for elimination of exogenous DNA, e.g., theuse of (iso)psoralen and UV irradiation (16, 22), provedunsuccessful.SSCP optimization. First, optimal SSCP conditions were

established. Gel matrixes that were evaluated included a 6%nondenaturing bis-acrylamide gel (30:0.8), a 6% HydrolinkLong Ranger gel, and a 0.5x MDE gel. These three gel typescontained the same amount of acrylamide, but the types andamounts of cross-linker differed. The standard bis-acrylamidegels did not produce SSCP patterns with high resolution evenwhen the percentage and/or ratio of bis/acrylamide waschanged. SSCP patterns with high resolution were observedwith both Hydrolink and MDE gels. However, color develop-ment during silver staining of Hydrolink gels was not repro-ducible. Therefore, further SSCP analyses were performedwith the MDE gel matrix.The effect of stabilizing ssDNA with glycerol was examined

by varying the amount of glycerol in the gel matrix. The bestresults were observed with the addition of 10% glycerol. TheSSCP gels could be reproducibly run at room temperaturewithout a temperature controller by simply using 0.75-mminstead of the standard 0.4-mm spacers.

Sensitivity of whole-cell-based PCR amplification and PCR-SSCP. A simple and rapid lysis procedure was used for bothgram-positive and gram-negative bacteria without the need forgenomic DNA isolation. Bacterial cells were lysed at 95°C withthe chelating agent Chelex 100 in the presence of the deter-gents sodium dodecyl sulfate, Nonidet P-40, and Tween 20 (9).The universal primers P11P and P13P used for PCR amplifi-cation were highly conserved, and amplification was observedfor all species tested. The sensitivity of PCR amplification withthe conserved primers was tested by amplifying E. coli and S.aureus cells for 35 cycles. The sensitivity obtained for PCRamplification on ethidium bromide-stained agarose gels was 30CFU for E. coli (Fig. 1) and 300 CFU for S. aureus. Thesedetection limits were obtained in combination with the pre-PCR sterilization protocol.

Silver staining is a promising alternative to radioactive SSCP(1, 17), but little is known about the sensitivity since thesensitivity of PCR-SSCP in terms of initial input of CFUbacteria rather than the amount of DNA in the acrylamide gelvisualized by silver staining is of interest for the application ofPCR-SSCP. Therefore, a serial dilution of E. coli ranging from103 to 3 CFU was tested with PCR-SSCP. The obtainedsensitivity of the system is approximately 30 CFU for E. coli(Fig. 2). In general, three or four bands of ssDNA were found,reflecting the fact that ssDNA may have multiple conforma-tions. These multiple conformations could be divided into oneor two stronger and one or two weaker SSCP bands.

TABLE 1. PCR-SSCP patterns with P11P-P13P obtained for 111bacterial isolates belonging to 40 species and 15 genera

Pattern no. Species

1 Acinetobacter calcoaceticus2 Bacteroides fragilis3 Citrobacter freundii

Citrobacter amalonaticus4 Clostridium difficile5 Clostridium bifermentans6 Clostridium butyricum7 Clostridium cadaveris8 Clostridium clostridioforme9 Clostridium histolyticum10 Clostridium innocuum11 Clostridium paraputnificum12 Clostridium perfringens13 Clostridium putnificum14 Clostridium sporogenes15 Escherichia coli

Shigella boydiiShigella dysenteriaeShigella flexneriSalmonella enterica subsp. enterica

16 Enterobacter aerogenes17 Enterobacter agglomerans18 Enterobacter cloacae19 Enterobacter gergoviae20 Enterobacter sakazakii21 Klebsiella oxytoca22 Klebsiella pneumoniae23 Listeria monocytogenes24 Listeria innocua25 Listeria ivanovii26 Morganella morganii27 Proteus mirabilis

Proteus vulgaris28 Pseudomonas aeruginosa29 Pseudomonas fluorescens30 Pseudomonas maltophilia31 Pseudomonas putida32 Staphylococcus aureus33 Staphylococcus epidernidis34 Group A hemolytic Streptococcus spp.

No. of strains

2231

10111111111181114345212374323231321242

PCR-SSCP patterns of a large panel of bacteria. PCR-SSCPis reported to be capable of detecting single-base mutations ina fragment of a few hundred base pairs and seems to be a

promising method for the differentiation of species. Because ofthe particular interest and expertise in our laboratory, we firstfocused on the applicability of PCR-SSCP for the detectionand identification of Clostridium and Listeria species (10, 24,26). Then we used 111 bacterial isolates belonging to 15 generaand 40 species to further test this technique (Table 1).Ten different strains of Clostridium difficile belonging to

different serotypes (serotypes A, B, C, D, F, G, H, I, K, and X)were analyzed by PCR-SSCP. These C. difficile strains showeddiscrete patterns with restriction fragment length polymor-phisms, restriction endonuclease analysis, and immunoblotting(26). All 10 C. difficile strains showed similar SSCP electro-phoresis patterns (Fig. 3), indicating that the region betweenthe primers is conserved for C. difficile. Ten other Clostridiumspecies were tested by PCR-SSCP to evaluate whether theamplified region is conserved at the genus or species level. Theelectrophoretic mobility patterns of these Clostridium speciesdiffered from the C. difficile patterns. All Clostridium speciesgave species-specific patterns. The observed PCR-SSCP pat-terns for Clostridium species were reproducible; assays per-

J. CLIN. MICROBIOL.

on May 22, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

PCR-SSCP FOR BACTERIAL IDENTIFICATION 3005

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20M

I~~~~~~~~~ ~~~~~~~~~~~ ~ ~~~~~~~~~~~~~~~~~~~~I7 1: C* * E. *

l l i ' , , a| t ~~~~~~~~~~~~~~~~~~~~~~~~~~~... .. ... *t"w*..'FN

MI 1 2 3 4 5 6 7 8 9 10 11 M

.:.

.F1

FIG. 3. SSCP analysis of Clostridium spp. after PCR amplificationof the bp 1175 to 1390 fragment of the 16S rRNA gene. Lanes 1 and20, control E. coli; lanes 2 to 11, C. difficile ATCC 9689 (serogroup A),ATCC 43593 (serogroup B), ATCC 43596 (serogroup C), ATCC43597 (serogroup D), ATCC 43598 (serogroup F), ATCC 43599(serogroup G), ATCC 43600 (serogroup H), ATCC 43601 (serogroupI), ATCC 43602 (serogroup K), and ATCC 43603 (serogroup X),respectively; lanes 12 to 19, C. clostridioforme, C. paraputnficum, C.putrificum, C. sporogenes, C. perfringens, C. innocuum, C. bifermentans,and C. butynicum, respectively; lane M, dsDNA molecular size markers(1,078, 872, and 603 bp).

formed on consecutive days gave identical electrophoreticpatterns.The 16S rRNA gene sequence for Listeria innocua exhibits

very high levels of relatedness with Listeria monocytogenes andListeria ivanovii sequences (99.2 and 98.4% homology, respec-tively) (8). By using the conserved 16S rRNA gene primers, L.innocua exhibits a 3-nucleotide difference with L. monocyto-genes and only a 2-nucleotide difference with L. ivanovii. Totest whether we can discriminate between these species, L.monocytogenes, L. innocua, and L. ivanovii strains were exam-ined for their electrophoretic mobility patterns in PCR-SSCP(Fig. 4). Although the observed differences were small, PCR-SSCP was capable of distinguishing L. innocua from the otherListeria species tested.

For the genus Enterobacter, the observed electrophoreticmobility patterns showed minor differences between E. agglo-merans, E. aerogenes, and E. cloacae (Fig. 5). E. cloacae couldbe differentiated from the last two species by the differentmobility of the lower ssDNA band, which was also the case forEnterobacter gergoviae (data not shown). Differences betweenthe species E. agglomerans and E. aerogenes were marginal andvisible only when the running time was sufficiently long.Enterobacter sakazakii showed a pattern totally different fromthose of the species described above.These data suggest species-specific patterns for Clostridium,

Listeria, and Enterobacter species. To evaluate the applicabilityof PCR-SSCP as a general tool for the identification ofbacteria, a large panel of bacterial isolates was tested (Table1). In general, patterns were specific at either the genus level orthe species level. Species-specific PCR-SSCP patterns wereobtained for Kiebsiella spp. (K oxytoca and K pneumoniae),Pseudomonas spp. (P. aeruginosa, P. fluorescens, P. maltophilia,and P. putida), and Staphylococcus spp. (S. aureus and S.epidermidis). Genus-specific patterns were observed forCitrobacter spp. (C. amalonaticus and C. freundii) and Proteusspp. (P. vulgaris and P. mirabilis). Acinetobacter calcoaceticus,Bacteroides fragilis, Morganella morganii, and group A hemo-lytic streptococci each gave conserved patterns different from

FIG. 4. PCR-SSCP with P11P-P13P for Listeria spp. Lanes 1 to 4,L. monocytogenes; lanes 5 to 7, L. innocua; lanes 8 to 10, L. ivanovii;lane 11, E. coli; lanes M, dsDNA molecular size markers (1,078, 872,and 603 bp).

those observed for the other species or genera. One exceptionwas found in the genus- or species-specific patterns; the closelyrelated E. coli, Shigella spp. (6), and Salmonella enterica subsp.enterica all gave similar PCR-SSCP patterns.

Since some of the different species or genera were not easilydistinguishable by the primer set P11P-P13P, another con-served primer set, ER10-ER11, was developed and tested forPCR-SSCP (Fig. 6). The amplified region is a 255-bp sequencelocated in the V2 region of the 16S rRNA gene. With thisprimer set, we were capable to discriminate between theclosely related E. coli, Shigella spp., and S. enterica subsp.enterica and species-specific patterns were observed for Proteusspp. (P. vulgaris and P. mirabilis) and Citrobacter spp. (C.amalonaticus and C. freundii), although the observed differ-ences for the latter genus are marginal. The combined use ofthe two conserved primer sets P11P-P13P and ER10-ER11

1 2 3 4 5 6 7 8 M

-b.

.~*

FIG. 5. PCR-SSCP with P11P-P13P for the genus Enterobacter.Lane 1, E. coli; lane 2, E. sakazakii; lanes 3 and 4, E. cloacae; lanes 5and 6, E. agglomerans; lanes 7 and 8, E. aerogenes; lane M, dsDNAmolecular size markers (1,078, 872, and 603 bp).

VOL. 32, 1994

on May 22, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

3006 WIDJOJOATMODJO ET AL.

5 6 7 8 9 10 11 12 13 14 15 16

FIG. 6. PCR-SSCP with primer pair ER10-ER11. Lane 1, P. vul-garis; lanes 2 to 4, P. mirabilis; lane 5, Shigella sonnei; lane 6, C.amalonaticus; lanes 7 to 9, C. freundii; lane 10, Shigellaflexneri; lane 11,Shigella dysenteniae; lanes 12 to 14, S. enterica subsp. entenca; lanes 15and 16, E. coli; lane M, dsDNA molecular size markers (1,353, 1,078,872, and 603 bp).

makes it possible to discriminate all tested bacteria at thespecies level, with the exception of the genus Shigella.

DISCUSSIONWe have developed a molecular biological method for

directly determining the identity of bacteria based on theprinciple of SSCP of a PCR-amplified DNA fragment. Thetarget DNA sequences were the bp 1175 to 1390 and bp 104 to358 regions of the 16S rRNA sequence by using conservedprimers. These primers flank a nonconserved region withspecies-specific sequences. The amplified PCR product con-tains species-specific sequences that can be subjected to SSCPto determine the identity of the bacteria, since the mobility ofthe ssDNA is sequence dependent. Nonradioactive detectionof the SSCP patterns was performed by a rapid and sensitivesilver staining method (3).

Since the use of conserved primers for PCR amplificationcan be hampered by contamination of PCR reagents withexogenous bacterial DNA (16, 21), we eliminated DNA con-

tamination by using a pre-PCR sterilization method withDNase I treatment of the PCR reagents prior to the additionof target DNA. This method was feasible only in combinationwith Super-Taq DNA polymerase and its buffer components.After DNase I sterilization, the obtained sensitivity with E. colicells of 30 CFU was comparable with the data of Greisen et al.(11), who amplified the bp 1170 to 1540 region of the 16SrRNA gene. The fact that we found satisfactory results ofeliminating exogenous DNA and inactivation of DNase I onlywith Super-Taq DNA polymerase corresponds with the find-ings of Bickler et al. (5). They found that thermolability ofDNase I is dependent on a high MgCl2 concentration, which isalso present in the PCR buffer for Super-Taq polymerase.

Gel electrophoresis conditions for the PCR-amplified targetsequence producing the best resolution included the 0.5 x

MDE gel matrix with addition of 10% glycerol. Under theseconditions electrophoresis resulted in three or four SSCPbands, indicating that ssDNA possessed multiple conforma-tions. These conformations could be divided into one or twomore pronounced bands and one or two less pronounced SSCPbands.PCR-SSCP with primer set P11P-P13P was tested on a panel

of 111 bacterial strains and showed conserved SSCP patterns ateither the genus or species level. The PCR-SSCP patternsobtained were genus specific for a few bacteria (e.g., Proteusspp.), but species specific for most bacteria (e.g., Pseudomonasspp., Clostidium spp., and Enterobacter spp.). C. difficile strainsbelonging to 10 different serotypes showed similar electro-phoretic mobility patterns, while other Clostridium speciesgave species-specific patterns. The closely related L. ivanoviiand L. innocua were distinguishable, even though only a2-nucleotide difference in the amplified region has been re-ported (8). The less pronounced SSCP bands were of impor-tance for the discrimination of species within the genusEnterobacter, since the pronounced SSCP bands showed indis-tinguishable band patterns. These results indicate the highdiscriminatory capacity of PCR-SSCP.The SSCP resolution was improved by the use of a second

set of conserved primers of the 16S rRNA gene; e.g., E. coliand Shigella and Salmonella spp. showed similar electro-phoretic mobility patterns with primer pair P11P-P13P butshowed different patterns with ER10-ER11. With the twoconserved primer sets, all 111 strains could be determined onthe basis of species-specific patterns, with only one exception,the genus Shigella, which was found to be conserved in bothregions.To overcome lane-to-lane and gel-to-gel differences, PCR-

SSCP resolution can be further improved by the addition of aninternal ssDNA marker for each sample lane. This makes itfeasible to precisely compare relative migration times. Also,simultaneous amplification of multiple conserved primer pairsin a multiplex PCR (4), based on polymorphism in severalregions of the 16S rRNA and/or 23S rRNA gene, mightsimplify the discrimination of a large panel of bacteria to thespecies level; PCR-SSCP with fluorochrome-labeled primers,e.g., fluorescein isothiocyanate, may be used in an alternativeSSCP detection system. SSCP can then be performed with anautomated sequencer that detects the fluorochrome-labeledDNA (15). One advantage of this system is that the sequenceris coupled to a computer which enables data management bycomputer. Recently, a paper described the use of fluorescein-labeled PCR-SSCP to distinguish between endomycorrhizalfungi with one fluorescein-labeled primer in the PCR (23).Here we have shown that PCR-SSCP is a promising fast

method for the identification of microorganisms. The use ofPCR with universal primers and SSCP patterns as an identifi-cation method can generally be applied to a wide range ofbacteria without the need of a large panel of probes. Since theidentification method is based on PCR, the obtained sensitivityis sufficient to detect low numbers of bacteria; the obtaineddetection limit by PCR-SSCP was 30 CFU for E. coli. The totaltime needed for PCR-SSCP is less than 20 h (2.5 h for PCRplus 15 h for SSCP plus 1.5 h for silver staining). PCR-SSCPseems a promising method for the rapid identification ofbacteria in usually sterile clinical sites, such as blood and thecerebrospinal fluid.

REFERENCES

1. Ainsworth, P. J., L. C. Surh, and M. B. Coulter-Mackie. 1991.Diagnostic single strand conformational polymorphism (SSCP): a

J. CLIN. MICROBIOL.

on May 22, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

PCR-SSCP FOR BACTERIAL IDENTIFICATION 3007

simplified non-radioisotopic method as applied to a Tay-Sachs Bivariant. Nucleic Acids Res. 19:405-406.

2. Barry, T., R Powell, and F. Gannon. 1990. A general method togenerate DNA probes for microorganisms. Bio,Technology 8:233-236.

3. Bassam, B. J., G. Caetano-Anolles, and P. M. Gresshoff. 1991. Fastand sensitive silver staining of DNA in polyacrylamide gels. Anal.Biochem. 196:80-83.

4. Bej, A. K., M. H. Mahbubani, R Miller, J. L. DiCesare, L. Hal,and R M. Atlas. 1990. Multiplex PCR amplification and immobi-lized capture probes for detection of bacterial pathogens andindicators in water. Mol. Cell. Probes 4:353-365.

5. Bickler, S. W., M. C. Heinrich, and G. C. Bagby. 1992. Magne-sium-dependent thermostability of DNase I. BioTechniques 13:64-66.

6. Brenner, D. J. 1992. Introduction to the family Enterobacteri-aceae, p. 2673-2695. In A. Balows, H. G. Truper, M. Dworkin, W.Harder, and K. H. Schleifer (ed.), The prokaryotes, vol. III, 2nded. Springer-Verlag, New York.

7. Chen, K., H. Neimark, P. Rumore, and C. Steinman. 1989. Broadrange DNA probes for detecting and amplifying eubacterialnucleic acids. FEMS Microbiol. Lett. 57:19-24.

8. Collins, M. D., S. Wallbanks, D. J. Lane, J. Shah, R Nietupski, J.Smida, M. Dorsch, and E. Stackebrandt. 1991. Phylogeneticanalysis of the genus Listeria based on reverse transcriptasesequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41:240-246.

9. De Lamballerie, X., C. Zandotti, C. Vignoli, C. Bollet, and P. DeMicco. 1992. A one-step microbial DNA extraction method using"Chelex 100" suitable for gene amplification. Res. Microbiol.143:785-790.

10. Fluit, A. C., R Torensma, M. J. C. Visser, C. J. M. Aarsman,M. J. J. G. Poppelier, B. H. I. Keller, P. Klapwjk, and J. Verhoef.1993. Rapid detection of Listeria monocytogenes in cheese with themagnetic immunopolymerase chain reaction assay. Appl. Environ.Microbiol. 59:1289-1293.

11. Greisen, K. M., M. Loeffelholz, A. Purohit, and D. Leong. 1994.PCR primers and probes for the 16S rRNA gene of most speciesof pathogenic bacteria, including bacteria found in cerebrospinalfluid. J. Clin. Microbiol. 32:335-351.

12. Gutell, R. R., N. Larsen, and C. R Woese. 1994. Lessons from anevolving rRNA: 16S and 23S rRNA structures from a comparativeperspective. Microbiol. Rev. 58:10-26.

13. Hayashi, K. 1992. PCR-SSCP: a method for detection of muta-tions. Genet. Anal. Tech. Appl. 9:73-79.

14. Jenssen, M. A., J. A. Webster, and N. Straus. 1993. Rapididentification of bacteria on the basis of polymerase chain reac-

tion-amplified ribosomal DNA spacer polymorphisms. Appl. En-viron. Microbiol. 59:945-952.

15. Makino, R, H. Yazyu, Y. Kishimoto, T. Sekiya, and K. Hayashi.1992. F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis.PCR Methods Appl. 2:10-13.

16. Meier, A., D. H. Persing, M. Finken, and E. Bottger. 1993.Elimination of contaminating DNA within polymerase chain re-action reagents: implications for a general approach to detectionof uncultured pathogens. J. Clin. Microbiol. 31:646-652.

17. Mohabeer, A. J., A. L. Hiti, and W. J. Martin. 1991. Non-radioactive single strand conformation polymorphism (SSCP)using the Pharmacia "Phast system." Nucleic Acids Res. 19:3154.

18. Muyzer, G., E. C. De Waal, and A. G. Uitterlinden. 1993. Profilingof complex microbial populations by denaturing gradient gelelectrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700.

19. Orita, M., H. Iwahana, K. Kanazawa, K. Hayashi, and T. Sekiya.1989. Detection of polymorphisms of human DNA by electro-phoresis as single-strand conformation polymorphisms. Proc. Natl.Acad. Sci. USA 86:2766-2770.

20. Orita, M., Y. Susuki, T. Sekiya, and K Hayashi. 1989. Rapid andsensitive detection of point mutations and DNA polymorphismsusing the polymerase chain reaction. Genomics 5:874-879.

21. Rand, K. H., and H. HoucL 1990. Taq polymerase containsbacterial DNA of unknown origin. Mol. Cell. Probes 4:445-450.

22. Rys, P. N., and D. H. Persing. 1993. Preventing false positives:quantitative evaluation of three protocols for inactivation ofpolymerase chain reaction amplification products. J. Clin. Micro-biol. 31:2356-2360.

23. Simon, L., R. C. Lvesque, and M. Lalonde. 1993. Identification ofendomycorrhizal fungi colonizing roots by fluorescent single-strand conformation polymorphism-polymerase chain reaction.Appl. Environ. Microbiol. 59:4211-4215.

24. Torensma, R, M. J. C. Visser, C. J. M. Aarsman, M. J. J. G.Poppelier, A. C. Fluit, and J. Verhoef. 1993. Monoclonal antibod-ies that react with live Listeria spp. Appl. Environ. Microbiol.59:2713-2716.

25. Wilson, K. H., R B. Blitchington, and R C. Greene. 1990.Amplification of bacterial 16S ribosomal DNA with polymerasechain reaction. J. Clin. Microbiol. 28:1942-1946.

26. Wolfhagen, M. J. H. M., A. C. Fluit, R Torensma, M. Jansze,A. F. A. Kuypers, E. A. E. Verhage, and J. Verhoef. 1993.Comparison of typing methods for Clostridium difficile isolates. J.Clin. Microbiol. 31:2208-2211.

VOL. 32, 1994

on May 22, 2020 by guest

http://jcm.asm

.org/D

ownloaded from