1. Introduction
 Fecal contamination of surface waters is a serious environmental
 problem that limits the use of water for various purposes, such as
 agriculture, aquaculture, source of drinking water and recreation
 (Simpson et al., 2002). The most widely used method for measuring
 fecal pollution of recreational water is to count fecal indicator
 bacteria, such as total coliforms, fecal coliforms, fecal enterococci
 and Escherichia coli. However, there are several limitations associated
 with traditional application of E. coli or fecal coliforms as indicators.
 First, standard methods involve the lengthy culturing time of at least
 18–24 h. Second, major microfloral populations of the human gut are
 not E. coli or fecal coliforms, but are obligate anaerobes, such as the
 classes Clostridia and Bacteroidetes in the phyla Firmicutes and
 Bacteroidetes/Chlorobi, respectively (Victor et al., 1996; Hayashi et
 ☆ Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank
 databases under the accession numbers GU130198 to GU130203.
 ⁎ Corresponding author. College of Public Health, Division of Environmental Health
 Sciences, The Ohio State University, Columbus, Ohio 43210, USA. Tel.: +1 614 292
 5546; fax: <PRESIDIO_ANONYMIZED_PHONE_NUMBER>.
 E-mail address: <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS> (J. Lee).
 0167-7012/$– see front matter © 2010 Elsevier B.V. All rights reserved.
 doi:10.1016/j.mimet.2010.07.012
 al., 2002; Hold et al., 2002).These are not easily cultivated (Eckburg et
 al., 2005).
 Recently, in order to overcome this hindrance, rapid detection
 methods, such as real-time PCR (Seurinck et al., 2005; Layton et al.,
 2006) or FISH (Collado and Sanz, 2007; Goh et al., 2009), have been
 developed by targeting 16S rRNA genes of significant fecal bacteria
 (e.g. genus Bacteroides (Okabe and Shimazu,2007). Oneadvantagefor
 using Bacteroides is that the genus hasa highdegreeofhost-specificity
 attributable to differences in source animal digestive systems, which
 enables Bacteroides to be used as an effective tool for microbial source
 tracking (MST) in water when molecular techniques are employed
 (Bernhard and Field, 2000; Simpson et al., 2004; Dick et al., 2005;
 Seurinck et al., 2005; Layton et al., 2006; Reischer et al., 2007).
 Even though many field tests have confirmed the efficacy of 16S
 rRNA gene-based molecular markers as human-specific or human
associated, false-positive and cross-amplification from other fecal
 sources, such as pig (Layton et al., 2006), dog (Ahmed et al., 2008),
 and cat (Kildare et al., 2007), have been reported. While some
 discrimination could be possible between homeothermic animal- and
 human-derived fecal contamination by 16S rRNA markers, it has been
 reported that 16S rRNA markers may not meet the standards as a
 useful tracking tool (McLain et al., 2009). The underlying problem is
312
 C.S. Lee, J. Lee / Journal of Microbiological Methods 82 (2010) 311–318
 the inherent limitation of the 16S rRNA gene as a marker for MST. 16S
 rRNA genes have a slow rate of evolutionary change and are thereby
 relatively conserved across related species which has resulted in few
 interspecies polymorphisms. Multiple ribosomal RNA (rrn) operon
 copies reside in a single bacterial genome (Acinas et al., 2004)
 contributing to the limited amount of interspecies polymorphism
 observed in some taxonomic groups (Boyer et al., 2001; Clarridge,
 2004).
 Furthermore, the genera Bacteroides and Prevotella are closely
 related to each other. The genus Prevotella is a moderately
 saccharolytic and bile-sensitive species, which once belonged to the
 genus Bacteroides (Shah and Collins, 1990). Many species of Prevotella
 have been isolated from human sources associated with the oral
 cavity (Sakamoto et al., 2004; Downes et al., 2005; Sakamoto et al.,
 2005a,b) and from tissues of the pelvic region, including the
 genitourinary tract, vagina and cervix (Brook and Frazier, 1997;
 Puapermpoonsiri et al., 1997; Mikamo et al., 1999; Fredricks et al.,
 2005; Hyman et al., 2005). All of these limitations of 16S rRNA-based
 methods make it difficult to discriminate target bacteria particularly
 when Bacteroides and Prevotella coexist in the contamination source.
 Recently, several housekeeping genes, such as rpoB, gyrB, elonga
tion factor Tu (EF-Tu), phosphoglycerate kinase (pgk), and heat shock
 protein (dnaK) genes, have been reported to evolve much faster than
 16S rRNA genes. Consequently they are used as alternative genetic
 markers providing better discrimination among closely related
 species (Volokhov et al., 2007). Eubacteria have two indispensable
 type II DNA topoisomerases; DNA gyrase encoded by gyrB and gyrA,
 and topoisomerase IV encoded by parE and parC (Huang, 1996).
 Amongthese genes, gyrB is a single-copy gene, present in all bacteria,
 whichencodestheB-subunitofDNAgyrase(topoisomerasetypeII).It
 maybeapotentiallyuseful geneforthedetectionandidentification of
 bacteria (Bhattacharya et al., 2003; Kawamura et al., 1999; Liu et al.,
 2003; Victor et al., 1996).
 Based on these advantages, this study focused on the gyrB gene
 marker in order to achieve higher specificity when targeting B. fragilis
 and to differentiate B. fragilis from other Bacteroides and Prevotella
 species. The performance of the real-time PCR system as a sensitive,
 specific and rapid tool for detection of recreational water contami
nation was investigated using human and cow fecal samples.
 2. Materials and methods
 2.1. Bacterial strains
 The type species and environmental isolates used in this study are
 listed on Table 1. The type strains were purchased from the American
 Type Culture Collection (Bacteroides fragilis ATCC 25285T, Bacteroides
 ovatus ATCC 8483T and Prevotella melaninogenica ATCC 25845T). B.
 fragilis ATCC 25285T and B. ovatus ATCC 8483T were propagated on B.
 fragilis bile-esculin agar (Oxyrase, Inc., Mansfield, OH) in GasPak jars
 (BBL Microbiology Systems, Cockeysville, MD) containing an Anaero
Pack system(Mitsubishi GasChemical, Tokyo,Japan) for 72 h at 37 °C.
 P. melaninogenica ATCC 25845T was grown at 37°C on Eggerth
 Gagnon (EG) agar (Eiken Chemical Co. Ltd., Tokyo, Japan) supple
Table 1
 Type strains used in this study.
 Strain
 environmental isolate, B. fragilis strains 1AL
 environmental isolate, B. fragilis strains 1B2
 environmental isolate, B. fragilis strains 1BL
 B. fragilis ATCC 25285T
 B. ovatus ATCC 8434T
 Accession no.
 gyrB
 GU130198
 GU130199
 GU130200
 CR626927
 AAXF02000049
 P. melaninogenica ATCC 25845T
 ACSI01000031
 16S rRNA
 GU130201
 GU130202
 GU130203
 CR626927
 AB050108
 AY323525
 mented with 5% (v/v) horse blood, 5 mg/l of haemin, and 0.1 mg/l of
 vitamin K1 in GasPak jars for 72 h at 37 °C. We also used three B.
 fragilis strains, 1AL, 1B2 and 1BL, which were isolated from
 environmental waterandidentifiedasB.fragilis with100%similarities
 of the 16S rRNA sequence of B. fragilis ATCC 25285T.
 2.2. Primer design based on gyrB gene
 Atotal of 322 gyrB sequences were retrieved from NCBI's GenBank
 database and aligned using Clustal X (Larkin et al., 2007). After the
 alignment, the sequences were manually searched for homologous
 regions specific for Bacteroides fragilis by using Bioedit (Hall, 1999).
 The selected regions were analyzed further to search homologous
 nucleotide sequences in the GenBank database by using the
 BLAST database search program (http://www.ncbi.nlm.nih.gov/
 tools/primer-blast/) and Probe Match application at the Ribosomal
 Database Project II (http://rdp.cme.msu.edu/probematch/). Several
 regions were selected for designing of primers and probe and each
 candidate was double-checked using the search programs, Primer
Blast and Probe Match, in order to minimize false cross-amplification
 with other bacteria. The Primer-Blast results were chosen because the
 NCBI database had more sequence information from environmental
 isolates and clones. Finally, one sequence subset was selected as a
 primer/probe set which showed the satisfying specificity for the B.
 fragilis. After obtaining the optimal sequence region of B. fragilis,
 forward andreverseprimers,and aminorgroovebinder(MGB)probe
 were determined by Primer Express® software (Applied Biosystems,
 Foster City, CA).
 2.3. Primer specificity tests with PCR
 In order to compare the specificity of the new gyrB-based primers,
 we conducted PCR amplification with a gyrB-based primer and two
 16S rRNA-based primer sets (qHS601F/qBac725R (Okabe et al., 2007)
 andg-Bfra-F/g-Bfra-R (Matsuki et al., 2002) with genomic DNAfrom3
 environmental isolates (1AL, 1B2, 1BL) and 3 type strains(B. fragilis, B.
 ovatus and P. melaninogenica). DNA templates were extracted from
 these 6 strains with a DNeasy Tissue Kit (Qiagen, Valencia, CA)
 following the manufacturer's instructions. Their concentrations were
 measuredwithNanoDrop(NanoDropTechnologies,Wilmington,DE).
 The DNA amount was adjusted as 10ng for template DNA per 1 PCR
 tubebecauseanamplificationreactionwouldbeconsiderablyaffected
 by the initial DNA concentration. PCR was conducted using a
 MultiGene Thermal Cycler (Labnet International, Inc., Edison, NJ) to
 generate a different optimal annealing point at the same time.
 Weused two reference 16S rRNA-based primers. For convenience
 of throughout this study, they will be referred to as Human-Bac1
 primer set for qHS601F/qBac725R and B.fragilis group-specific primer
 set for g-Bfra-F/g-Bfra-R, respectively. We compared our newly
 designed gyrB-based primers with these two references. The
 Human-Bac1 primer set had an optimal annealing temperature of
 62 °C and the B. fragilis group-specific primers had a low annealing
 temperature of 50 °C. The optimal annealing temperature for the gyrB
 primers was 58 °C and the amplification condition was the same with
 the exception of the annealing temperature. PCR was carried out by
 incubation for 3 min at 94 °C, 30 cycles of denaturation at 94 °C for
 1 min, annealing at each appropriate temperature for 30 s, extension
 at 72 °Cfor1 min,finalextensionat72 °Cfor10 minwitheachprimer
 set. After PCR amplification, aliquots (5 μl) of the PCR products were
 analyzed by electrophoresis on a 1.0% agarose gel stained with 0.1 μg/
 ml ethidium bromide in TBE buffer (89 mM Tris–borate, 2 mM EDTA)
 at 100 V for 40min. The bands were documented with a Bio-Rad
 Quantity One Gel Doc system (Bio-Rad, Hercules, CA) with a 1 kb
 (Invitrogen, Carlsbad, CA) DNA ladder as molecular weight marker.
C.S. Lee, J. Lee / Journal of Microbiological Methods 82 (2010) 311–318
 313
 2.4. Sequencing and phylogenetic analysis
 Excess primers and dNTPs were removed from the remaining PCR
 products using a QIAquick PCR purification kit (Qiagen, Valencia, CA).
 The amplified DNA products were sequenced at the Plant-Microbe
 Genomics Facility at The Ohio State University (http://pmgf.biosci.
 ohio-state.edu/) by an ABI Prism 3730 DNA analyzer (Applied
 Biosystems). Multiple alignments of the DNA sequences allowed
 phylogenetic comparisons between the Bacteroides and Prevotella
 species with the gyrB and 16s rRNA genes. Reference sequences of
 Bacteroides and Prevotella were retrieved from GenBank database,
 aligned by Clustal X and trimmed by Bioedit which allowed the
 construction of two phylogenetic trees based on 16S rRNA and gyrB
 genesusingMEGA(Tamuraetal.,2007).Theneighborjoiningmethod
 with Kimura 2 parameter model (Saitou and Nei, 1987), Maximum
likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge and
 Farris, 1969) were used to draw trees. A bootstrap analysis was
 performed to assess the confidence limits of the branching with 1000
 replicates. Only values greater than 50 were included in the results.
 2.5. Standard curves and comparison of gyrB-based and 16S rRNA-based
 primers by TaqMan real-time PCR
 With the newly designed gyrB-based primers, real-time PCR was
 carried out to obtain standard curves of B. fragilis. The Human Bac1
 primerset wastestedasa16SrRNA-basedreferenceprimerset.TheB.
 fragilis group-specific primer set, which is another 16S rRNA-based
 reference, was not originally designed for the TaqMan system, but for
 the SYBR probe system, so it could not be directly comparable with
 our primer set. Serial dilutions of B. fragilis were prepared by spiking
 cultured cells into 1 ml of sterile phosphate buffered saline (PBS;
 0.14 M NaCl, 2.7 mM KCl, 10.1mM Na2HPO4, 1.8mM KH2PO4 [pH
 7.6]) (Sigma-Aldrich, St. Louis, MO). Ten μl of the well mixed solution
 of B. fragilis was used to count colony forming units (CFU) after
 anaerobic culture on BBE plates and the 990 μl of the remaining
 solution was used for DNA extraction using a DNeasy Tissue kit
 (Qiagen, Valencia, CA). The extracted DNA was used for real-time PCR
 to quantify B. fragilis. Real-time PCR was performed and a standard
 curve was derived by plotting threshold cycles (CT) against the log
transformed cell counts (CFU/PCR).
 An ABI 48-well StepOneTM Real-Time System (Applied Biosys
tems) was used with a total 25 μl mixture which consisted of 5 μlof
 template DNA, 12.5 μl of TaqMan universal PCR master mix (PCR
 buffer, deoxynucleoside triphosphates, AmpliTaq Gold polymerase,
 internal reference signal 6-carboxy-x-rhodamine [ROX], Amp Erase
 uracil N-glycosylase [UNG], MgCl2; Applied Biosystems), 500 nM of
 each primer, and 250nM of each probe labeled with 6-carboxy
 f
 luorescein (FAM). A mixture of all PCR regents without any template
 DNA was used as a negative control for each PCR reaction. Thermal
 cycling consisted of an initial cycle of 50 °C for 2 min (activation of the
 UNG) and 95°C for 10min (activation of the AmpliTaq Gold DNA
 polymerase), followed by 40 cycles of denaturation at 95 °C for 15 s
 and annealing and extension at 60 °C for 1 min. For the Bacteroides
Prevotella-specific primer set, PCR reactions were carried out by
 incubation for 2 min at 50 °C and for 10 min at 95°C, followed by
 40 cycles of denaturation at 95 °C for 15 s and annealing and
 extension at 62 °C for 1 min.
 2.6. End-point and real-time PCR assays for cow, pig, dog, and human
 fecal samples
 In order to check the host-specificity of the newly designed primer
 set, we testedthe specificity of all three primersets withfecal samples
 from cow, pig, dog, and human. Individual fresh fecal samples were
 collected from apparently healthy human and animal sources. Cow
 and pig feces were obtained from the Waterman farm (Dairy Science
 Center, TheOhioStateUniversity,Columbus,Ohio).Dogsampleswere
 obtained from local pet owners and represented several different
 breeds. All fecal samples were collected using sterile swabs (BBL
 CultureSwab; Becton Dickinson, Sparks, MD) and immediately
 transported to the laboratory on ice. For DNA extraction, 10 ml of
 sterile PBS was added directly into the swabs and vortexted. The fecal
 suspension (1 ml) was transferred into a sterile microcentrifugal tube
 and its weight was measured by subtracting the post-centrifugation
 weight from the pre-centrifugation weight. The centrifugation was
 performed at 10,000 g for 1 min at 4 °C. With the known concentra
tion of fecal suspensions, all the fecal samples were adjusted to the
 same amount of 5mg fecal pellet by transferring appropriate volume
 into the tube. DNA was extracted using the QIAmp DNAStool Mini Kit
 (Qiagen) for end-point and real-time PCR assays with Human Bac1
 and gyrB-based primers. The concentration of the DNA was deter
mined from the final elute of 200 μl from the DNA extraction with a
 NanoDrop system (NanoDrop Technologies).
 3. Result
 3.1. Designing of gyrB-based primers for B. fragilis
 Analysis of the 322 gyrB sequences of Bacteroides and Prevotella in
 the GenBank database allowed the identification of B. fragilis-specific
 regions and the design of the primer set (Fig. 1 and Table 2). One
 primer set covering only 54-bp size was composed of a forward
 primer (Bf904F), a reverse primer (Bf958R), and the TaqMan probe
 (Bf923MGB). An additional reverse primer was designed (Bf1272R)
 because it could generate enough size of sequences (368-bp) for
 phylogenetic analysis.
 Multiple alignment analysis showed that our reverse primer,
 Bf958R, supported higher specificity for B. fragilis than Bf904F. We
 evaluated the specificity of the newly designed gyrB-based primer
 (Bf904F/Bf958R), 16S rRNA-basedHumanBac1(qHS601F/qBac725R),
 and B. fragilis group-specific (g-Bfra-F/g-Bfra-R) primer sets using B.
 fragilis, other Bacteroides (B. ovatus) and Prevotella (P. melaninogenica)
 by end-point PCR. The results showed a distinction in the amplicons
 bythethreeprimersetsintargetingB. fragilis (Fig. 2). As expected, we
 obtained 54-bp size gyrB gene products from both environmental B.
 fragilis isolates and type strain of B. fragilis (ATCC 25285T), and
 negative results from B. ovatus and P. melaninogenica when using
 Bf904F/Bf958R (Fig. 2A). The 150-bp size 16S rRNA gene regions by
 qHS601F/qBac725R were amplified in all of environmental B. fragilis
 isolates and type strains of B. fragilis, B. ovatus and P. melaninogenica
 (Fig. 2B). The 495-bp size 16S rRNA gene region by g-Bfra-F/g-Bfra-R
 was amplified only in environmental B. fragilis isolates and type
 strains of B. fragilis and B. ovatus, but not from P. melaninogenica
 (Fig. 2C). The PCR results confirm that our gyrB-based primer, Bf904F/
 Bf958R, can discriminate between B. fragilis and other closely related
 species, such as B. ovatus and P. melaninogenica.
 3.2. Sequencing and phylogenetic analysis
 The PCR products produced by our gyrB-based primer set and one
 16S rRNA-based Human Bac1 primer set (qHS601F/qBac725R) were
 further investigated by sequence analysis. The other 16S rRNA-based
 B. fragilis group-specific primer (g-Bfra-F/g-Bfra-R) was not compared
 for this purpose because it had a significantly different amplicon size
 (495-bp) from the amplicons produced by the gyrB-based (54-bp)
 and HumanBac1(150-bp) primers. The sequences (Fig. 3) confirmed
 again that new proposed primer set could amplify only the gyrB gene
 of B. fragilis. Although we obtained the PCR products from B. fragilis by
 using the Bf904F/Bf958R, we could not obtain sequencing data
 because their sequences were too short to analyze sequencing. In
 order to overcome this limitation, another reverse primer (BF1272R)
 was used to amplify N100 bp of PCR amplicons for sequence analysis.
C.S.Lee, J.Lee/ JournalofMicrobiologicalMethods82(2010)311
Fig.1.MultiplealignmentsoftheB. fragilis-specificprimers,Bf904F,Bf958R,Bf1275R,andtheTaqManprobeBf923MGBfortheB. fragilisgyrBsequenceincomparisonwiththe
 homologousgyrBsequencesofotherBacteroidesandPrevotella
 Analyzed sequences were added up with other 16S rRNA and gyrB
 gene sequences from GenBank and EMBL databases to construct
 phylogenetic dendrograms.
 Bacteroides 16S rRNA sequences are as follows: B. acidifaciens
 (AB021164), B. caccae (X83951), B. capillosus (AY136666), B. cellulosi
lyticus (AJ583243), B. dorei (AB242142), B. eggerthii (AB050107), B.
 f
 inegoldii (AB222699), B. fragilis (CR626927), B. gallinarum (AB253732),
 B. graminisolvens (AB363973), B. helcogenes (AB200227), B. intestinalis
 (AB214328), B.massiliensis (AY126616), B. nordii (AY608697), B.ovatus
 (AB050108), B. pectinophilus (DQ497993), B. plebeius (AB200217), B.
 pyogenes(AB200229),B.salyersiae(AY608696),B.stercoris(X83953),B.
 tectus (AB200228), B. thetaiotaomicron (AE015928), B. uniformis
 (AB050110), B. vulgatus (CP000139), B. xylanisolvens (AM230650).
 Prevotella 16S rRNA sequences are as follows: P. bryantii (AJ006457), P.
 buccae (L16477), P. copri (AB064923), P. heparinolytica (L16487), P.
 micans (AF481228), P. nanceiensis (AY957555), P. oralis (AY323522),
 P. oris (L16474), P. paludivivens (AB078827), P. ruminicola (L16482), P.
 shahii (AB108825).
 Bacteroides gyrBsequencesareasfollows:B.caccae(AAVM02000009),
 B. capillosus (AAXG02000004), B. cellulosilyticus (ACCH01000128),
 B. dorei (DS995534), B. eggerthii (ABVO01000051), B. finegoldii
 (ABXI02000076), B. fragilis (CR626927), B. intestinalis (ABJL02000007),
 B. ovatus (AAXF02000049), B. pectinophilus (ABVQ01000037), B. plebeius
 (ABQC02000004), B. stercoris (ABFZ02000020), B. thetaiotaomicron
 (AE015928), B. uniformis (AAYH02000048), B. vulgatus (CP000139),
 Prevotella gyrB sequences are as follows: P. copri (ACBX01000202), P.
 melaninogenica (ACSI01000031), P. oralis (AB048191), P. tannerae
 (ACIJ02000017).
 Twophylogenetic trees were created with the available sequences
 of 16S rRNA and gyrB gene from GenBank and EMBL database, so not
 all the bacterial species could be equally entered in each tree. Thus, a
 simple and direct comparison of the genetic distances of the two
 phylogenetic trees was not feasible. However, the following observa
tions were made. First, the rate of genetic divergence of the sequence
 amplified by the gyrB-based primer set differed greatly from that of
 the 16S rRNA-based primer set. For example, compared with the
 sequence amplified by the 16S rRNA-based primers, the sequence
 amplifiedbythegyrBprimersetshoweda4-to10-foldincreaseinthe
 length of branches between the closely related species of Bacteroides
 and Prevotella. Moreover, the topology of the phylogenetic tree based
 on the 16S rRNA sequence was quite different from the tree based on
 the gyrB sequence. It demonstrated that 8 Bacteroides species (B.
 acidifaciens, B. nordii, B. fragilis, B. salyersiae, B. thetaiotaomicron, B.
 xylanisolvens, B. finegoldii and B. ovatus) were clustered into one group
 in the 16S rRNA-based phylogenetic tree (Fig. 3A), but they were
 totally separated into each cluster in the gyrB gene-base tree (Fig. 3B).
 Secondly, there was a difference in the classification of the target
 bacteria between the sequences amplified by the 2 primers. For
 instance, even though the 16S rRNA sequences of 3 environmental
 isolates, 1AL, 1B2 and 1BL, were 100% identical to the type strain of B.
 fragilis, their intrageneric relatedness with other species of Bacteroides
 was too close to be positioned solely with the B. fragilis species
 (Fig. 3A). In contrast, with the amplification by the gyrB gene, these 3
 isolates were clearly clustered only with the B. fragilis species (Fig. 3B).
 3.3. Performance of real-time PCR with new primers and probe for
 B. fragilis
 Aseries of experiments were conducted with B. fragilis to compare
 the results obtained using gyrB-based (Bf904F/Bf923MGB/Bf958R)
 and the 16S rRNA-based primers and probe (qHS601F/qHS624MGB/
 qBac725R)(Fig. 4). For this, 10-fold serial dilutions of known B. fragilis
 cells were prepared and CT values were checked with the 48-well
 StepOne™Real-TimeSystem (Applied Biosystems) using each primer
 set. The TaqMan PCR reagent contains a passive reference dye as an
 internal reference to which the reporter dye was normalized during
 data analysis. This allows correction of fluctuations in fluorescence
 due to changes in concentration and volume of the reaction mixture.
 Table2
 Nucleotidesequencesandtargetsofprimersandprobesusedforreal-timePCRassayinthisstudy.
 Name Sequence Gene Target Reference
 Bf904F 5′-GGCGGTCTTCCGGGTAAA-3′ gyrB B. fragilis Thisstudy
 Bf923MGB 5′-TGGCCGACTGCTC-3′ gyrB B. fragilis Thisstudy
 Bf1272R 5′-TGGCATATAGCGGAAGAAAAAAG-3′ gyrB B. fragilis Thisstudy
 Bf958R 5′-CACACTTCTGCGGGTCTTTGT-3′ gyrB B. fragilis Thisstudy
 qHS601F 5′-GTTGTGAAAGTTTGCGGCTCA-3′ 16SrRNA B. fragiliscluster Okabeetal.(2007)
 qHS624MGB 5′-CGTAAAATTGCAGTTGA-3′ 16SrRNA B. fragiliscluster Okabeetal.(2007)
 qBac725R 5′-CAATCGGAGTTCTTCGTGATATCTA-3′ 16SrRNA Bacteroides–Prevotella BernardandField(2000)
 g-Bfra-F 5′-ATAGCCTTTCGAAAGRAAGAT-3′ 16SrRNA B. fragilisgroup Matsukietal.(2002)
 g-Bfra-R 5′-CCAGTATCAACTGCAATTTTA-3′ 16SrRNA B. fragilisgroup
TheCTvalueisthecycleatwhichastatisticallysignificantincreasein
 ΔRnwasfirstdetectedassociatedwithexponential increaseofPCR
 product.Linearregressionanalysesofthereal-timePCRresultsofthe
 gyrBandthe16SrRNAampliconsproducedstandardcurveswith
 similar slopes (−3.367,−3.332)andgoodcorrelationcoefficients
 (r2=0.998, 0.997), butdifferenty-intercepts(41.89, 38.99) inthe
 rageof1.1×102to1.1×107(CFU/PCR).
 3.4.End-pointandreal-timePCRassays forcow,pig,dog,andhuman
 fecalsamples
 Host-specificitywas testedwithhumanandotheranimal fecal
 samplesusingend-pointPCR(Table3).TheextractedDNAconcen
trations were 2.8±0.6ng/μl (cow, n=10), 4.0±0.9ng/μl (pig,
 n=10), 3.4±0.9ng/μl (dog, n=10), and3.6±0.5ng/μl (human,
 n=10).Allthethreeprimersetsshowed100%positivePCRreaction
 with thehumansamples. However, somecross-amplificationwas
 observed fromnon-human fecal samples.With theHumanBac1
 (qHS601F/qBac725R)primerset,positivereactionswereobservedin
 70%pig,40%cow,and30%dogsamples.WiththeB. fragilisgroup
specificprimer(g-Bfra-F/g-Bfra-R),positivereactionswereobtained
 from50%pig,30%cow,and10%dogsamples.WiththegyrB-based
 primerset(Bf904F/BF958R),positivereactionswereobservedinonly
 10%pigsamplesandnocross-amplificationwasobtainedfromallthe
 cowand dog samples. This result indicates that the gyrB-based
 B. fragilis-specific primer has superior specificity against human
originatedfecalcontamination.
 Thesamesetoffecalsampleswastestedwithreal-timePCRassayfor
 validatinghuman-specificfecalcontamination.WiththeuseofHuman
 Bac1, theaverageCtvalueswere18.61±1.23(human),21.33±2.00
 (pig), 24.75±1.40 (cow), and 28.07±4.89 (dog) and theywere
 equivalentto1.4±0.8×1010,3.8±4.6×109,2.2±1.7×108,and2.1±
 3.2×108CFU/goffeces.Thisconversionwasbaseduponthestandard
 curve(Fig.4B)andthedilutionfactors.Thesameprocedurewasused
 withthegyrB-basedprimerset.TheaverageCtvalueswere27.86±0.66
 (human),35.64±0.68(pig),35.91±0.80(cow)and35.85±0.56(dog)
 and theywere equivalent to 1.3±0.7×108, 4.6±4.5×105, 4.0±
 4.0×105,2.1±2.8×105CFUB.fragilis/goffeces,respectively.
 4.Discussion
 The B. fragilis-specific detection by real-time PCR allows the
 quantificationofB. fragilis,whichisasignificantandrepresentative
 human-specificfecal indicator.Thisisthefirststudy1)todevelopa
 primersetthattargetsthegyrBgeneofB.fragilisasanewfecalsource
 marker;and2)optimizetheTaqManreal-timePCRsystemforrapid
 detectionofahuman-specificfecalsourceinrecreationalwater.We
 also investigatedthespecificityof ournewlydesignedprimersby
 comparingpreviouslyknown16SrRNA-basedprimersets.Byshifting
 thetargetmarker from16SrRNAtothegyrBgene,weareableto
 proposeanalternativeprimer systemwhich isveryspecific toB.
 fragilisasarepresentativespecies inBacteroides.Variouscandidate
 markersweredeterminedaftermultiplealignmentsof gyrBgenes
 fromtheavailabledatabaseand twoprimer setswere chosen. A
 primerset,Bf904F/Bf958R,wasusedtoamplifythe904–958regionof
 thegyrBgeneinareal-timePCRandcomparedwiththe16SrRNA
based referenceprimer set (HumanBac1). Theother primer set,
 Bf904F/Bf1272R,wasusedtoamplifythe904–1272regionofthegyrB
 geneinordertoanalyzesequencesforphylogeneticinvestigation.The
 newgyrB-basedprimer showedhigh specificity toB. fragilis and
 negativereactiontoB. ovatusandP.melaninogenica,whicharethe
 mostcloselyrelatedtoB. fragilis.
 TheothergyrB-basedprimerset(368-bpcoveringBf904F/Bf1272R
 primerset)wasmoreusefulforanalyzingsequencesofPCRamplicons
 andconstructingphylogenetictreesbecausetheampliconsproduced
 bytheBf904F/Bf958Rprimer setwasnot longenoughtoprovide
 sufficientinformationonsequences.PCRampliconsweresequenced
 andanalyzedwith16SrRNAfragmentsandthegyrB-basedphyloge
netic tree showed that it had greater resolution enabling the
 generationofamoredivergeddendrogram.Eachphylogenetictree
 wasconstructedwithpreviouslyknownsequences intheGenBank
 database. The twoconstructeddendrogramsdemonstrate that the
 gyrB-basedprimersetdifferentiatedB. fragilisfromotherBacteroides
 andPrevotellaspecies, but the16SrRNA-basedprimersetdidnot.
 OnlyB. fragiliswasamplifiedby thegyrB-basedprimer, but all B.
 fragilis, B. ovatusandP.melaninogenicawereamplifiedbythe16S
 rRNA-basedprimers.Thismayserveasapossibleexplanationtowhy
 manyfieldstudiesofMSTtargetingBacteroidesperformedinconsis
tentlyinthepast(Laytonetal.,2006;Kildareetal.,2007;Ahmedetal.,
 2008;McLainetal.,2009).
 ThenewlydevelopedTaqManprimer/probesystemforreal-time
 PCRwasoptimizedtoquantifyB. fragilisinwaterbyspikingaknown
 numberofbacterialcells.Thereal-timePCRresultsdemonstratethat
 thegyrB-basedprimersetcanbeusedtoquantifyB. fragiliswiththe
 detectionlimitof110CFU/reaction.ThegyrB-basedreal-timePCRhad
 aslightlylesssensitivitythantheoneusing16SrRNA-basedprimer
 set. The difference in y-intercepts of the calibration curveswas
 approximately3cyclesbetweenthegyrB-basedandthe16SrRNA
basedsystem,whichindicatesthat3morecyclesareneededtoyield
 thesameCTvalues.Itisequivalenttoapproximatelya1logdifference
 in colony forming unit (CFU). The differences in real-time PCR
 conditionsofthegyrB-basedandthe16SrRNA-basedprimers,suchas
 themelting point, mayhave affected the sensitivity of the PCR
 amplification,whichisexpressedasCTvalues.Additionally,16SrRNA
 genesaremorelikelytobeamplifiedthangyrBgenesbecauseatleast
 6 copies of 16S rRNAgene are contained in one B. fragilis cell
 (Klappenbachet al., 2001), butonlyonegyrBgeneexistsper cell
 (Huang,1996).Thus, fromthesamenumberofB. fragilis, inherently
 differentamountofgyrBand16SrRNAgeneswouldbeobtainedin
 theextractedDNA.Thereby, it isexpectedtoobservedifferencesin
 they-interceptsof thestandardcurvesaswellasdifferencesinthe
 sensitivityofdetectionbyreal-timePCR.
 Duringour preliminary study,we investigated the gyrB-based
 assaysystemwithrecreationalwaterinordertocheckwhetherthe
 detectionisfeasibleandwhetherthedetectionlimitisaffectedbyPCR
 inhibitorspossiblypresent inrecreationalwater.Thereal-timePCR
 Fig.2.Specificityofthenewlydesignedprimers.PCRproductsbynewgyrB-basedprimer(A)andtwo16SrRNA-basedprimers(B,C).
 315 C.S.Lee, J.Lee/ JournalofMicrobiologicalMethods82(2010)311
testforB. fragilispresent intherecreationalwaterandPBSresulted
 very similar CT values (data not shown). Statistically significant
 differencebetweenthePBSandtherecreationalwatersamplewas
 notobservedinthereal-timePCRdetectionofB. fragilis(datanot
 shown).Thisindicatedthattheenvironmentalwatermatrixdidnot
 affectthereal-timePCR.
 Anessential traitof ageneticmarker toserveasastableand
 library-independent MST tool is its temporal and geographical
 Fig.3.Comparisonofphylogenetictreesbasedon16SrRNAgeneandthegyrBgeneforthegenusBacteroidesandPrevotella.
 316 C.S.Lee, J.Lee/ JournalofMicrobiologicalMethods82(2010)311
humanandthe16SrRNA-basedhuman-specificmarkersmayhave
 thepossibilityofcross-amplificationswithswinefeces.
 Wehavedescribedarapidmonitoringsystemforahuman-specific
 fecal indicator, B. fragilis, based on the gyrB genemarker for
 indispensable type II DNA topoisomerases inbacteria. The gyrB
basedprimerhadsufficientresolutionpowertoamplifyonlyB.fragilis
 species and gyrB genemarker canbe used to designate and/or
 differentiatebacterial speciesorsubspecieswhicharespecific toa
 host.Duringthisstudy,wetestedourprimersetwithfecalsamples
 fromanimal andhumansources. ThegyrB-basedprimer set could
 differentiatehuman fecal samples fromanimal fecal samples and
 producepositiveresultsforB. fragilis.16SrRNAgeneisstillusefulfor
 groupingandclusteringinphylogeneticstudy.However, switching
 thetargetgenefrom16SrRNAtogyrBmaybeveryuseful formore
 accurate identification, classification, and phylogenetic sorting of
 bacterial lineages (YamamotoandHarayama, 1995; LaDucet al.,
 2004). Thismethod could serve as a complementary tool for
 overcoming the limitation of 16S rRNA-basedMSTmethods in
 delineatingabilityamongclosely relatedbacteriaoriginated from
 humansources.Inaddition,itcanbeappliedtoothermoleculartools
 that involveprimers andprobes, suchasmicroarray, T-RFLP, and
 othernucleotide-basedmethods