question:stage.set_background("tilewall") sprite = codesters.Sprite("person6", 0, -50) password = " " #my_display = codesters.Display(my_var, x, y) my_display = codesters.Display(password, -200, 150) website = sprite.ask("What website is this password for?") password = website[0] + website[-1] my_display.update(password) purpose = sprite.ask("What do you use this website for?") purpose_words_list = purpose.split() print(purpose_words_list) for word in purpose_words_list: print(word[0]) password += word[0] sprite.say(password, 1) password += str(len(purpose_words_list)) my_display.update(password) sibs = sprite.ask("How many siblings do you have?") password += sibs my_display.update(password) Request more user input and store it in a new variable.

question:Rewrite this email: Good day, This is Mr. Stupart. I am writing to inform you that your student has been late to Chorus on multiple occasions. Today, your child was 13 minutes late to class without a pass. Please speak with your student about getting to class on time. Any further tardies will result in a conquence. Thank you for your attention. Mr. Stupart

question:lūdzu pastāsti par sociālās palīdzības ietekmi uz bezdarbu, tā palielinās vai samazinās?

question: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