research article rapid and sensitive pcr-dipstick dna...

Research ArticleRapid and Sensitive PCR-Dipstick DNA Chromatographyfor Multiplex Analysis of the Oral Microbiota

Lingyang Tian12 Takuichi Sato1 Kousuke Niwa34 Mitsuo Kawase3

Anne C R Tanner5 and Nobuhiro Takahashi1

1 Division of Oral Ecology and Biochemistry Tohoku University Graduate School of Dentistry Sendai 980-8575 Japan2Department of Prosthodontics West China Hospital of Stomatology Sichuan University No 14 Section 3 South Renmin RoadChengdu 610041 China

3 Tohoku University Graduate School of Biomedical Engineering Sendai 980-8579 Japan4 Future Technology Management Center Corporate RampD NGK Insulators Mizuho Nagoya 467-8530 Japan5Department of Microbiology The Forsyth Institute Cambridge MA 02142 USA

Correspondence should be addressed to Takuichi Sato takmtohokuacjp

Received 1 July 2014 Revised 3 September 2014 Accepted 8 September 2014 Published 17 November 2014

Academic Editor Kazuhiko Nakano

Copyright copy 2014 Lingyang Tian et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A complex of species has been associated with dental caries under the ecological hypothesis This study aimed to develop a rapidsensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaquebacteria Parallel oligonucleotides were immobilized on a dipstick strip formultiplex analysis of target DNA sequences of the caries-associated bacteria Streptococcus mutans Streptococcus sobrinus Scardovia wiggsiae Actinomyces species and Veillonella parvulaStreptavidin-coated blue-colored latexmicrospheres were to generate signal TargetDNAampliconswith an oligonucleotide-taggedterminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridizedwith complementary oligonucleotides on the stripThe accumulation of captured latex beads on the test and control lines producedblue bands enabling visual detection with the naked eye The PCR-dipstick DNA chromatography detected quantities as low as100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis dependingon the target bacterial species Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samplesThe assay time was less than 3 hThe semiquantificationprocedure revealed the relative amounts of each test species in dental plaque samples indicating that this disposable device hasgreat potential in analysis of microbial composition in the oral cavity and intestinal tract as well as in point-of-care diagnosis ofmicrobiota-associated diseases

1 Introduction

With increasing appreciation of the role of bacterial commu-nities in the etiology of dental caries [1] information aboutthe qualitative and quantitative composition and changesin oral biofilms are needed to understand the infectionassociated with disease initiation and progression [2ndash4] Thedevelopment of a rapid simple accurate point-of-care tech-niques for oral microbiota profiling particularly the detec-tion and quantification of oral pathogens has thus become amatter of urgency The advent of polymerase chain reaction(PCR) initiated a revolution in clinical microbiology from

traditional approaches such as microscopy culture antigendetection and immunoserology to molecular techniquespredominated by nucleic acid amplification- (NAA-) basedmethods [5 6] PCR amplification provides good specificitysensitivity reproducibility of results and a basis for the devel-opment of assays that exploit differences in DNA sequencesSubstantial efforts have beenmade to optimize PCRmethods[7] and different protocols including standardized directPCRmultiplex PCR and themore sensitive nested PCR havebeen developed and applied to oral microbiology [8ndash14]

Simplicity is another advantage of PCR NAA-basedtechniques avoid the need for in vitro cultivation and shorten

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 180323 10 pageshttpdxdoiorg1011552014180323

2 BioMed Research International

the time to detect species from days to several hoursHowever conventional post-PCR analysis using gel elec-trophoresis with ethidium bromide staining is hazardouslabor-intensive and time-consuming and may not be suf-ficiently specific and sensitive Advanced probe or capturehybridization methods developed in partial response tothese drawbacks still require multiple procedures includingheat denaturation hybridization and washing and expensiveinstrumentation [15] DNA biosensors provide an alterna-tive approach to simplifying post-PCR analysis with greatlyenhanced detectability specificity and reproducibility Thegeneral principle of DNA biosensors involves the immobi-lization of a DNA probe on the surface of the sensor withhybridization and detection by electrochemical [16] optical[17] or gravimetric means [18] PCR can be carried outwith primers with bulky substituents bound to their 51015840 or31015840 ends which is useful for the design of DNA biosensorsSpecific oligonucleotide sequences or immunologic sub-stances (eg hapten-antibody and hapten-protein) that offerextremely strong affinity to capture DNA amplicons havebeen investigated as DNA probe candidates More recentlyprogress on DNA biosensors has enabled visual detection ofDNA by employing gold nanoparticles [19ndash22] and coloredpolystyrene beads [23] as signal generators Quantitativedata can be recorded by intensity readout or by additionalcoupling with an enzyme [20] fluorescent dye [24 25] orradioactive conjugates [26] Further developments of DNAbiosensors have exploited more powerful strategies includingquantitative real-time PCR [27ndash31] DNA microarrays [32]and surface plasmon resonance [33 34] These techniqueshave emerged as novel quantitative methods in microbiologybut are expensive and require trained laboratory technicians

A growing number of DNA biosensors have beendesigned for point-of-care diagnosis in the form of lateralflow (dipstick) nucleic acid test strips [19ndash24 26 36] Paper-based microfluidic bioassay appears to meet all the keystandards of a point-of-care diagnostic device [37] that isa sensitive and accurate assay that requires no complexequipment reagents or power sources and preferably amethod that is rapid small disposable and easy to use andtransport

This study aimed to develop a combination of PCR anddipstick-type DNA biosensors into one system as a rapidsensitive and visible multiplex analysis device to profilethe oral microbiota targeting the caries-associated bacte-ria Streptococcus mutans Streptococcus sobrinus Scardoviawiggsiae Actinomyces species and Veillonella parvula Thesebacteria have been known as crucial bacteria in the oralcavity as followsMutans streptococci in particular Smutansand S sobrinus have been thought to be associated withdental caries since they have frequently been isolated fromdental plaque biofilm where they produce large amountsof acids and extracellular polysaccharides which promotedental caries It has been reported that S wiggsiae has astrong association with early childhood caries in the USA[38] Actinomyces species are predominantly detected fromdental plaque biofilm periodotitis lesions and root surfacecaries [39] Veillonella species such as Veillonella parvulaare one of the indigenous bacteria of the oral cavity (dental

plaque biofilm and tongue coating) and the species obtaintheir energy by fermenting organic acids for example lactate[40] This metabolism has the potential to remove a potentdental-caries producing acid Most of the current DNAbiosensors utilize streptavidin-biotin interaction as the DNAcapture mechanism allowing only single detection of a targetdsDNA in one test To simultaneously test for several bacterialspecies different oligonucleotide strands were immobilizedin parallel on one strip In addition streptavidin-coatedcolored latex microspheres were used as reporters to givechromatic signals Semiquantitative information based onvisual intensity was attempted by a standardized 10-foldserial-dilution without any specialized reagents or instru-mentation

2 Materials and Methods

21 Bacterial Strains Five bacterial strains Streptococcusmutans NCTC 10449 Streptococcus sobrinus 6715 Scardoviawiggsiae F0424 Actinomyces oris WVU627 and Veillonellaparvula ATCC 10790 were cultured on CDC anaerobic 5sheep blood agar (BD Franklin Lakes NJ) plates and wereincubated at 37∘C for 3 days in an anaerobic glove box (ModelAZ-Hard Hirasawa Tokyo Japan) under an atmosphere of80 N

2 10 H

2 and 10 CO

2

22 Subjects and Sample Collection After obtaininginformed consent supragingival plaque (15mg) fromcaries-free enamel surfaces was sampled from 16 healthysubjects (mean age 318 plusmn 89 years range 23ndash54 years)The dentition (dental caries) status was recorded using theDMFT index (decayed missing and filled teeth) and thirdmolars were excluded No subjects had taken antibiotics for3 months prior to sampling Plaque samples were collectedusing sterile toothpicks and stored in 15mL sampling tubesat minus20∘C before analysis

23 DNA Extraction Genomic DNA was extracted fromdental plaque or 3-day-old bacterial cultures using the Insta-Gene Matrix Kit (Bio-Rad Laboratories Richmond CA)according to the manufacturerrsquos instructions

24 Polymerase Chain Reaction Direct PCR was performedusing the tagged species-specific primers listed in Table 1Each PCR mixture comprised 21 120583L of either plaque sam-ple DNA or bacterial genomic DNA 25120583L of Taq DNApolymerase (HotStar Taq PLUS Master Mix Qiagen GmbHHilden Germany) and 4 120583L of primer mixture PCR amplifi-cation was conducted in a PCRThermal Cycler MP (TaKaRaBiomedicals Ohtsu Shiga Japan) or an iCycler (Bio-RadLaboratories) programmed for 5min at 95∘C initial heatactivation and 30 cycles of 1min at 95∘C for denaturation1min at appropriate temperatures (in Table 1) for annealingand 15min at 72∘C for extension followed by 10min at 72∘Cfor final extension

25 Dipstick DNA Chromatography Dipstick DNA stripsand reagents were obtained commercially (TBA Co Sendai

BioMed Research International 3

Table1PC

Rprim

ersa

ndcond

ition

s

Bacterialstrain

Targeted

gene

Prim

ername

Sequ

ence

(51015840-31015840)

Size

(bp)

Ann

ealin

gtemperature

(∘ C)

Smutan

s16SrRNAgenes

Sm1(575F)

Tag1-X

-GGTC

AGGAAAG

TCTG

GAG

TAAAAG

GCT

A282

56Sm

2(856R)

Biotin-G

CGGTA

GCT

CCGGCA

CTAAG

CC

Ssobrinus

16SrRNAgenes

SobF

(54F

)Tag2-X-

CGGAC

TTGCT

CCAG

TGTT

ACTA

A546

56SobR

(599R)

Biotin-G

CCTT

TAAC

TTCA

GAC

TTAC

Swiggsiae

16SrRNAgenes

Scar

448F

Tag3-X-

GTG

GAC

TTTA

TCAAT

AAG

C172

51Scar

619R

Biotin-C

TACC

GTT

AAG

CAGTA

AG

Actin

omyces

species

16SrRNAgenes

AorF

Tag4-X-

GGCT

GCG

ATAC

CGTG

AGG

104

56Ao

rRBiotin-TCT

GCG

ATTA

CTAG

CGAC

TCC

Vparvula

rpoB

genes

Vp2696F

Tag5-X-

GAAG

CATT

GGAAG

CGAAAG

TTTC

G623

57Vp

3318R

Biotin-G

TGTA

ACAAG

GGAG

TACG

GAC

CAc

tinom

ycesspeciesincludesA

orisA

naeslu

ndiiandA

odontolyticus

ldquoXrdquorepresentsldquospacer

C3rdquoTh

eSpacerC

3[th

ree-carbon

spacer(CH

2)3usingPh

osph

oram

ideC

3spacer

(GlenRe

searchSterling

VA)]isinserted

betweenTags

1(to

5)andar

espectiveforwardprim

er[35]


Absorptionpart

Control line

S sobrinus (cTag 2 line 2)

Actinomyces species(cTag 4 line 4)

S mutans (cTag1 line 1)

S wiggsiae (cTag 3 line 3)

V parvula (cTag 5 line 5)

Marker (red line)

(a)

Biotin StretpavidinTarget DNA amplicon

Biotin

Control line

Flow direction

Latex bead

Absorption part

TagX

V p

arvu

la

S m

utan

s

S so

brin

us

S w

iggs

iae

Test lines 1ndash5

cTag 1 cTag 2 cTag 3 cTag 4 cTag 5

Actin

omyc

essp

ecie

s

(b)

Figure 1 (a) (b) Schematic diagram of dipstick DNA chromatography Tag = oligonucleotide strand cTag = complementary oligonucleotidestrand

Japan) As shown in Table 1 for each pair of primers the 51015840terminus (of forward primer) was tagged with five differentoligonucleotides and the 51015840 terminus (of reverse primer) wasbiotinylated Because the Spacer C3 [three-carbon spacer(CH2)3 using Phosphoramide C3 spacer (Glen Research

Sterling VA)] (representing ldquoXrdquo in Figure 1(b) and Table 1)is inserted between Tags 1 (to 5) and a respective forward

primer the DNA synthesis by Taq DNA polymerase termi-nates at the insertion site [35] Blue-colored latex particlescoated with streptavidin were linked to biotinylated terminalamplicons through a streptavidin-biotin interaction Dipstickstrips (25mmtimes 45mm)weremanufactured by immobilizingfive complementary oligonucleotides to specifically recognizethe PCR amplicons through hybridization with 51015840 terminus


Figure 2 Dipstick-DNA chromatography 1 negative control 2ndash6 single detection of Streptococcus mutans Streptococcus sobrinusScardoviawiggsiaeActinomyces oris andVeillonella parvula respec-tively 7 multiplex detection

tags On the strip 12 test lines in total were available and thefirst 5 in order were selected in the present study A biotin-immobilized flow control line was set up at the end of thestrip (Figure 1) One PCR-amplified product was diluted intoa total volume of 30 120583L and mixed with 10 120583L of eluent (con-taining detergents blocking agents PBS and salts solution)and 2120583L of streptavidin-coated blue latex suspension (bothof them were supplied by TBA Co) A DNA strip was theninserted into the mixture A visible blue test line (05mmwidth 12mmapart from each other) would appear where thecomplementary oligonucleotide was immobilized indicatingthe presence of target DNA sequences

26 Sensitivity of Dipstick DNA Chromatography To validatethe sensitivity of dipstick DNA chromatography aliquotscontaining 10 ng 1 ng 100 pg 10 pg or 1 pg of DNA amplifiedby PCR from five bacterial strains were prepared and assayedin parallel The detection limit of dipstick DNA chromatog-raphy for each bacterial strain was determined by the visualinspection

27 Specificity and Sensitivity of PCR-Dipstick DNA Chro-matography The specificity of PCR-dipstick DNA chro-matography was confirmed under the following conditions(1) oligonucleotide-free primers (2) biotin-free primers and(3) no target DNA template Cross reaction of the primerswith other bacteria was previously examined by in vitroexperiments using representative oral bacteria [10 38 41 42]In addition in order to confirm the specificity of primersin silico BLAST searches were performed in this studythrough the website of the National Center for BiotechnologyInformation We found that the primers of S sobrinus cross-reacted with Streptococcus downei and those of Actinomycesspecies (Actinomyces oris Actinomyces naeslundii and Acti-nomyces odontolyticus) cross-reacted with Micromonosporapeucetia however S downei and M peucetia have not beenreported to be isolated from the human oral cavity

In order to assess the assay sensitivity serial 10-fold dilutions of purified DNA from each bacterial strain

(range 10 ng to 1 fg) were amplified by PCR and dividedinto 2 equal parts One part was used for dipstick DNAchromatography and the detection limit was determinedTheother part was assayed with 1 agarose gel electrophoresis(High Strength Analytical Grade Agarose Bio-Rad Labora-tories) as follows PCR products (10 120583L each) were separatedon 1 agarose gels in Tris-borate EDTA buffer (100mMTris 90mM borate 1mM EDTA pH 84) stained withethidium bromide photographed under UV light A 100-bpDNA Ladder (Invitrogen Corp Carlsbad CA) was used as amolecular size marker

28 Semiquantification Assay Procedure for Plaque SamplesSemiquantitative analysis of clinical samples requires rigor-ous optimization and standardization DNA was extractedfrom dental plaque samples and amplified by PCR foreach test species (Table 2) PCR-amplified DNA was 10-folddiluted from the original concentration to a 104 multiple anda 1 120583L aliquot at the same dilution factor for each species wasmixed and detected by dipstick DNA chromatography Thedetection limit of each bacterial species in dental plaque wasobtained from a serial chromatogram and the relative con-centration of each bacterial species was estimated based onthe dilution fold for the detection limit with correction usingthe relative sensitivity of PCR-dipstickDNAchromatographyas described above

3 Results and Discussion

31 Validation Principle of Dipstick DNA ChromatographyIn the reaction mixture the biotin-labeled PCR ampliconimmediately attached to the streptavidin-coated blue latexparticles due to the strong biotin-streptavidin affinity to forma hybrid When the tip of the dipstick strip was insertedinto the mixture PCR amplicon-blue latex hybrids movedup through the strip by capillary action and were capturedby tag-complement oligonucleotide fixed as test line on thestrip resulting in accumulation of blue latex microspheres toinduce a visible blue test line The flow control line on theother tip of strip was set up to confirm the proper function ofthe assay and also to capture excess latex particles indicatingthat the amount of latex particles was capable of catchingall the DNA amplicons Different DNA amplicons couldbe detected either alone within 5min or simultaneously inone assay after 5ndash10min with no cross reactions (Figure 2)thus confirming that this method is applicable for multiplexanalysis

The development of dipstick biosensors has entered intoa more advanced stage which is focusing on enhancing themultiplexing capabilities Biosensors with two or more testlines were subsequently proposed to simultaneously detectseveral alleles for genotyping of a biallelic polymorphism[43ndash45] These biosensors involved either primer extension(PEXT) or oligonucleotide ligation reactions (OLR) whichusually required an additional step in the assays A methodthat detects the dsDNA could maintain the simplicity of adipstick DNA biosensor Such DNA biosensors have beenreported by utilizing oligonucleotides to bind PCR amplicons


Table 2 Procedure for semiquantification of plaque samples

Procedure Item Amount (volume)Plaque sampling Dental plaque 15mgDNA extraction InstaGene Matrix Kit 300 120583L

PCR

DNA extraction used for PCR 21 120583LPrimer F (tagged) (5120583M) 2 120583LPrimer R (biotinylated) (5 120583M) 2 120583LHot StarTaq PLUS Master Mix 25 120583L

Dipstick DNA chromatography

DNA amplified by PCR 1 120583LDeionized water 29 120583LEluent (supplied by TBA Co containing detergents blockingagents PBS and salts solution) 10120583L

Streptavidin-coated blue latex suspension (supplied by TBA Co) 2 120583L

Figure 3 Detection limit of dipstick DNA chromatographyAmount of each target DNA amplicon detected on strips 1ndash5 10 ng1 ng 100 pg 10 pg and 1 pg

with reporters and streptavidin-biotin as capture mecha-nism which allow only one target dsDNA amplicon to bedetected [19] In contrast in the present study five differentoligonucleotides complementary to corresponding tags weremounted on one strip thus enabling multiplex detection ofdsDNA sequences in one assay

32 Sensitivity of Dipstick DNA Chromatography The detec-tion limit of dipstick DNA chromatography was evaluatedusing purified DNA amplified from each bacterial strainso as to exclude the influence of the PCR procedure onsensitivity (Figure 3) For all five species PCR product couldbe detected at levels as low as 100 pg Intensitywas observed tobe proportional to DNA concentration When the amount ofDNA product was tested in the useful analytical range belowthe saturation concentration intensity provided quantitativeinformation on target DNA [19] which laid the foundationfor the following semiquantification analysis

33 Specificity and Sensitivity of PCR-Dipstick DNA Chro-matography In this study five test lines generated by targetDNA hybrids appeared where expected When five target

Figure 4 Specificity of PCR-dipstick DNA chromatography 1 5target amplicons tagged by oligonucleotides at 51015840 ends and biotin at31015840 ends 2 target amplicons tagged by oligonucleotides only 3 targetamplicons tagged by biotin only 4 PCR performed with no targetDNA templates

DNA amplicons were detected in one assay no smear orfalse positive lines were observed on the strip The resultsof specificity assessment are presented in Figure 4 Analysisperformed with oligonucleotide-free or biotin-free primersor in the absence of target DNA templates gave no positivesignals Under these three circumstances the streptavidin-coated blue latex particles could not link to the oligonu-cleotide detectors to generate blue signal in the test zoneand were finally captured in the control line The specificityof dipstick DNA chromatography using the same biosensorswas also confirmed by another project in which 176 bacterialstrains were detected [46] In addition no cross reactionswith other bacteria were observed in silico (in this study) orin vitro [10 38 41 42] thus supporting the high specificity ofthis method


Table 3 Detection limit of PCR-dipstick DNA chromatography and PCR-agarose gel electrophoresis

100 pg 10 pg 1 pg 100 fg 10 fg 1 fg

S mutans Dip + + + + + +Aga + + + minus minus minus

S sobrinus Dip + + minus minus minus minus

Aga + minus minus minus minus minus

S wiggsiae Dip + + + minus minus minus

Aga + + minus minus minus minus

Actinomyces species Dip + + + minus minus minus


V parvula Dip + + + + minus minus

Aga + + + minus minus minus

Figure 5 Semiquantitative chromatogram of sample 1 1 120583L-aliquotfor each specific PCR amplification was detected Dilution factorsfor strips 1ndash5 original concentration 10-fold 102-fold 103-fold and104-fold respectively

A comparison of the detection sensitivity obtained usingthe PCR-dipstick DNA chromatography and PCR-agarosegel electrophoresis is shown in Table 3 Although there weredifferences among the five bacterial species PCR-dipstickDNA chromatography showed at least 10-fold higher sen-sitivity than agarose gel electrophoresis demonstrating theadvantages of PCR-dipstick DNA chromatography

34 Semiquantification of Dental Plaque Samples The resultsfrom PCR-dipstick DNA chromatography using purifiedbacterial DNA showed that the sensitivity of this methoddiffered between bacterial species (Table 3) As dipstick DNAchromatography exhibited the same detection limit for allfive species (Figure 3) there were differences in detectionsensitivity that could only be derived from PCR amplificationefficiency The relative PCR amplification efficiencies (Er) ofS mutans S sobrinus S wiggsiaeA oris andV parvulawereestimated to be 1 10minus4 10minus3 10minus3 and 10minus2 respectively fromTable 3 Thus relative concentrations of bacteria (Cr) couldbe calculated by multiplying dilution fold for detection limit

(D) and dividing by relative PCR efficiency (Er) using thefollowing equation

Cr =DEr

(1)

Cr is relative concentration of a given bacteria in dentalplaqueD is dilution fold of detection limit Er is relative PCRamplification efficiency

An additional issue is that the color density may dependon the amount of DNA molecules as well as other factorsincluding the amount of latex microspheres and oligonu-cleotides impregnated on the test lines however once asystem is standardized quantification could still be achievedby rigorously controlling the whole procedure

PCR-dipstick DNA chromatography assay was applied to16 supragingival plaque samples A chromatogram (Figure 5)was obtained for each sample Actinomyces species wasdetected in all 16 samples with unanimously high DNAconcentrations S mutans andV parvula presented in 15 of 16samples While frequently detected these two species variedsignificantly in amount among samples S sobrinus wasdetected in only 1 sample with the lowest detection frequencyfollowed by S wiggsiae with 4 positive samples out of 16 Thepositive species for each samplewere enumeratedAt least 2 ofthe 5 species tested were detected in the supragingival plaquesamples

Semiquantitative analysis was carried out based on theabove calculation (Table 4) From relative concentration ofeach bacterial strain the composition of the oral microbiotacould be investigated simply and rapidly Taking sample 1as an example (Figure 5) S sobrinus and S wiggsiae wereabsent Among the 3 positively detected species V parvulapredominated over Actinomyces species and S mutans Therelative concentrations of S mutans Actinomyces speciesand V parvula were estimated to be 1 105 105 Actinomycesspecies and V parvula were observed to be the predominantbacteria in all tested samples S mutans exhibited higherdetection frequency but lower proportion among the wholeplaque bacteria when compared with previous reports [1247] which could be attributed to the low detection limit ofPCR-dipstick DNA chromatography and the use of differentprimersTherewere no significant differences among subjects


Table4Semiquantificatio

nof

fiveg

iven

bacteriain

16supragingivalplaqu

esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43


with low moderate and high DMFT (Table 4) in the detec-tion frequencies (and amounts) of the five bacteria

4 Conclusions

A disposable paper-based PCR-dipstick DNA chromatogra-phy method was developed for simple rapid and multiplexanalysis of bacterial profiles in microbiota by impregnatingseveral oligonucleotides on one strip to specifically recognizetarget DNA sequences Semiquantitative information on theamount of target bacterial DNA in dental plaque can beobtained from dilution fold for detection limit of eachbacterial species with correction using relative PCR efficiencyof each bacterial speciesThe turnaround time from samplingto semiquantification requires only 3 h and this time couldbe further compressed by refining the PCR method Thismethod can be applied to analysis of microbiota such asin the oral cavity and intestinal tract and to point-of-carediagnosis of microbiota-associated diseases such as dentalcaries and periodontitis This universally designed methodmay be modified for a variety of purposes

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

This study was supported in part by Grants-in-Aid forScientific Research (24390511 25462945 25463237 2567077725861785 and 26462869) from the Japan Society for thePromotion of Science

References

[1] K A Brogden J M Guthmiller and C E Taylor ldquoHumanpolymicrobial infectionsrdquo The Lancet vol 365 no 9455 pp253ndash255 2005

[2] W F Liljemark and C Bloomquist ldquoHuman oral microbialecology and dental caries and periodontal diseasesrdquo CriticalReviews in Oral Biology and Medicine vol 7 no 2 pp 180ndash1981996

[3] N Takahashi ldquoMicrobial ecosystem in the oral cavitymetabolicdiversity in an ecological niche and its relationship with oraldiseasesrdquo International Congress Series vol 1284 pp 103ndash1122005

[4] H F Jenkinson and R J Lamont ldquoOral microbial communitiesin sickness and in healthrdquo Trends inMicrobiology vol 13 no 12pp 589ndash595 2005

[5] NArnheim andH Erlich ldquoPolymerase chain reaction strategyrdquoAnnual Review of Biochemistry vol 61 pp 131ndash156 1992

[6] A CWhelen andDH Persing ldquoThe role of nucleic acid ampli-fication and detection in the clinical microbiology laboratoryrdquoAnnual Review of Microbiology vol 50 no 1 pp 349ndash373 1996

[7] R A Gibbs ldquoPolymerase chain reaction techniquesrdquo CurrentOpinion in Biotechnology vol 2 no 1 pp 69ndash75 1991

[8] T Igarashi A Yamamoto and N Goto ldquoPCR for detectionand identification of Streptococcus sobrinusrdquo Journal of MedicalMicrobiology vol 49 no 12 pp 1069ndash1074 2000

[9] T Oho Y Yamashita Y Shimazaki M Kushiyama and TKoga ldquoSimple and rapid detection of Streptococcus mutans andStreptococcus sobrinus in human saliva by polymerase chainreactionrdquo Oral Microbiology and Immunology vol 15 no 4 pp258ndash262 2000

[10] S Rupf KMerte K Eschrich L Stosser and S Kneist ldquoPeroxi-dase reaction as a parameter for discrimination of Streptococcusmutans and Streptococcus sobrinusrdquo Caries Research vol 35 no4 pp 258ndash264 2001

[11] E J Leys A L Griffen S J Strong and P A Fuerst ldquoDetectionand strain identification of Actinobacillus actinomycetemcomi-tans by nested PCRrdquo Journal of Clinical Microbiology vol 32no 5 pp 1288ndash1294 1994

[12] T Sato J Matsuyama T Kumagai et al ldquoNested PCR fordetection of mutans streptococci in dental plaquerdquo Letters inApplied Microbiology vol 37 no 1 pp 66ndash69 2003

[13] G Mayanagi T Sato H Shimauchi and N Takahashi ldquoDetec-tion frequency of periodontitis-associated bacteria by poly-merase chain reaction in subgingival and supragingival plaqueof periodontitis and healthy subjectsrdquo Oral Microbiology andImmunology vol 19 no 6 pp 379ndash385 2004

[14] S D Tran and J D Rudney ldquoImproved multiplex PCR usingconserved and species-specific 16S rRNA gene primers forsimultaneous detection of Actinobacillus actinomycetemcomi-tans Bacteroides forsythus and Porphyromonas gingivalisrdquoJournal of Clinical Microbiology vol 37 no 11 pp 3504ndash35081999

[15] J G Lazar ldquoAdvancedmethods in PCR product detectionrdquo PCRMethods and Applications vol 4 no 1 pp S1ndashS14 1994

[16] T G DrummondMGHill and J K Barton ldquoElectrochemicalDNA sensorsrdquo Nature Biotechnology vol 21 no 10 pp 1192ndash1199 2003

[17] M Minunni S Tombelli E Mariotti andMMascini ldquoBiosen-sors as new analytical tool for detection of GeneticallyModifiedOrganisms (GMOs)rdquo Fresenius Journal of Analytical Chemistryvol 369 no 7-8 pp 589ndash593 2001

[18] S Tombelli M Minunni and M Mascini ldquoPiezoelectricbiosensors strategies for coupling nucleic acids to piezoelectricdevicesrdquoMethods vol 37 no 1 pp 48ndash56 2005

[19] K Glynou P C Ioannou T K Christopoulos and V Syri-opoulou ldquoOligonucleotide-functionalized gold nanoparticlesas probes in a dry-reagent strip biosensor for DNA analysis byhybridizationrdquo Analytical Chemistry vol 75 no 16 pp 4155ndash4160 2003

[20] X Mao Y Ma A Zhang L Zhang L Zeng and G LiuldquoDisposable nucleic acid biosensors based on gold nanoparticleprobes and lateral flow striprdquo Analytical Chemistry vol 81 no4 pp 1660ndash1668 2009

[21] A Chua C Y YeanM Ravichandran B Lim and P Lalitha ldquoArapid DNA biosensor for the molecular diagnosis of infectiousdiseaserdquo Biosensors and Bioelectronics vol 26 no 9 pp 3825ndash3831 2011

[22] G Y Ang C Y Yu andC Y Yean ldquoAmbient temperature detec-tion of PCR amplicons with a novel sequence-specific nucleicacid lateral flow biosensorrdquo Biosensors and Bioelectronics vol38 no 1 pp 151ndash156 2012

[23] D P Kalogianni I K Litos T K Christopoulos and P C Ioan-nou ldquoDipstick-type biosensor for visual detection of DNAwitholigonucleotide-decorated colored polystyrene microspheres asreportersrdquo Biosensors and Bioelectronics vol 24 no 6 pp 1811ndash1815 2009


[24] L Wang W Chen W Ma et al ldquoFluorescent strip sensor forrapid determination of toxinsrdquo Chemical Communications vol47 no 5 pp 1574ndash1576 2011

[25] D Pyo and J Yoo ldquoNew trends in fluorescence immunochro-matographyrdquo Journal of Immunoassay and Immunochemistryvol 33 no 2 pp 203ndash222 2012

[26] P Corstjens M Zuiderwijk A Brink et al ldquoUse of up-converting phosphor reporters in lateral-flow assays to detectspecific nucleic acid sequences a rapid sensitive DNA testto identify human papillomavirus type 16 infectionrdquo ClinicalChemistry vol 47 no 10 pp 1885ndash1893 2001

[27] M Kubista J M Andrade M Bengtsson et al ldquoThe real-timepolymerase chain reactionrdquoMolecular Aspects of Medicine vol27 no 2-3 pp 95ndash125 2006

[28] B Kaltenboeck and C M Wang ldquoAdvances in real-time PCRapplication to clinical laboratory diagnosticsrdquo Advances inClinical Chemistry vol 40 no 4 pp 219ndash259 2005

[29] M J Espy J R Uhl LM Sloan et al ldquoReal-time PCR in clinicalmicrobiology applications for a routine laboratory testingrdquoClinical Microbiology Reviews vol 19 no 1 pp 165ndash256 2006

[30] Y Abiko T Sato G Mayanagi and N Takahashi ldquoProfilingof subgingival plaque biofilm microflora from periodontallyhealthy subjects and from subjects with periodontitis usingquantitative real-time PCRrdquo Journal of Periodontal Researchvol 45 no 3 pp 389ndash395 2010

[31] M Yamaura T Sato S Echigo and N Takahashi ldquoQuantifi-cation and detection of bacteria from postoperative maxillarycyst by polymerase chain reactionrdquo Oral Microbiology andImmunology vol 20 no 6 pp 333ndash338 2005

[32] P O Brown and D Botstein ldquoExploring the new world of thegenome with DNA microarraysrdquo Nature Genetics vol 21 no 1pp 33ndash37 1999

[33] Z Altintas Y Uludag Y Gurbuz and I Tothill ldquoDevelopmentof surface chemistry for surface plasmon resonance basedsensors for the detection of proteins and DNA moleculesrdquoAnalytica Chimica Acta vol 712 pp 138ndash144 2012

[34] J M McDonnell ldquoSurface plasmon resonance towards anunderstanding of the mechanisms of biological molecularrecognitionrdquo Current Opinion in Chemical Biology vol 5 no5 pp 572ndash577 2001

[35] KNiwa AOribe HOkumura et al ldquoTaghybridization-basedsensitive detection of polymerase chain reaction productsrdquoAnalytical Biochemistry vol 464 pp 12ndash16 2014

[36] C-Z Li K Vandenberg S Prabhulkar et al ldquoPaper basedpoint-of-care testing disc for multiplex whole cell bacteriaanalysisrdquo Biosensors amp Bioelectronics vol 26 no 11 pp 4342ndash4348 2011

[37] A K Yetisen M S Akram and C R Lowe ldquoPaper-basedmicrofluidic point-of-care diagnostic devicesrdquo Lab on a Chipvol 13 no 12 pp 2210ndash2251 2013

[38] A C R Tanner R L Kent P L Holgerson et al ldquoMicrobiota ofsevere early childhood caries before and after therapyrdquo Journalof Dental Research vol 90 no 11 pp 1298ndash1305 2011

[39] K Hashimoto T Sato H Shimauchi and N TakahashildquoProfiling of dental plaque microflora on root caries lesions andthe protein-denaturing activity of these bacteriardquoTheAmericanJournal of Dentistry vol 24 no 5 pp 295ndash299 2011

[40] J Washio Y Shimada M Yamada R Sakamaki and N Taka-hashi ldquoHydrogen sulfide production by oralVeillonella effect ofpH and lactaterdquo Applied and Environmental Microbiology vol80 no 14 pp 4184ndash4188 2014

[41] S Periasamy N I Chalmers L Du-Thumm and P E Kolen-brander ldquoFusobacterium nucleatumATCC 10953 requiresActi-nomyces naeslundii ATCC 43146 for growth on saliva in athree-species community that includes Streptococcus oralis 34rdquoApplied and Environmental Microbiology vol 75 no 10 pp3250ndash3257 2009

[42] E Igarashi A Kamaguchi M Fujita H Miyakawa and FNakazawa ldquoIdentification of oral species of the genusVeillonellaby polymerase chain reactionrdquoOralMicrobiology and Immunol-ogy vol 24 no 4 pp 310ndash313 2009

[43] D K Toubanaki T K Christopoulos P C Ioannou and C SFlordellis ldquoIdentification of single-nucleotide polymorphismsby the oligonucleotide ligation reaction a DNA biosensorfor simultaneous visual detection of both allelesrdquo AnalyticalChemistry vol 81 no 1 pp 218ndash224 2009

[44] I K Litos P C Ioannou T K Christopoulos J Traeger-Synodinos and E Kanavakis ldquoMultianalyte dipstick-typenanoparticle-based DNA biosensor for visual genotyping ofsingle-nucleotide polymorphismsrdquo Biosensors and Bioelectron-ics vol 24 no 10 pp 3135ndash3139 2009

[45] D S Elenis P C Ioannou and T K Christopoulos ldquoAnanoparticle-based sensor for visual detection of multiplemutationsrdquo Nanotechnology vol 22 no 15 Article ID 1555019 pages 2011

[46] M Hayashi T Natori S Kubota-Hayashi et al ldquoA new protocolto detect multiple foodborne pathogens with PCR dipstickDNA chromatography after a six-hour enrichment culturein a broad-range food pathogen enrichment brothrdquo BioMedResearch International vol 2013 Article ID 295050 10 pages2013

[47] S Hata HHata HMiyasawa-Hori A Kudo andHMayanagildquoQuantitative detection of Streptococcus mutans in the dentalplaque of Japanese preschool children by real-time PCRrdquoLettersin Applied Microbiology vol 42 no 2 pp 127ndash131 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the time to detect species from days to several hoursHowever conventional post-PCR analysis using gel elec-trophoresis with ethidium bromide staining is hazardouslabor-intensive and time-consuming and may not be suf-ficiently specific and sensitive Advanced probe or capturehybridization methods developed in partial response tothese drawbacks still require multiple procedures includingheat denaturation hybridization and washing and expensiveinstrumentation [15] DNA biosensors provide an alterna-tive approach to simplifying post-PCR analysis with greatlyenhanced detectability specificity and reproducibility Thegeneral principle of DNA biosensors involves the immobi-lization of a DNA probe on the surface of the sensor withhybridization and detection by electrochemical [16] optical[17] or gravimetric means [18] PCR can be carried outwith primers with bulky substituents bound to their 51015840 or31015840 ends which is useful for the design of DNA biosensorsSpecific oligonucleotide sequences or immunologic sub-stances (eg hapten-antibody and hapten-protein) that offerextremely strong affinity to capture DNA amplicons havebeen investigated as DNA probe candidates More recentlyprogress on DNA biosensors has enabled visual detection ofDNA by employing gold nanoparticles [19ndash22] and coloredpolystyrene beads [23] as signal generators Quantitativedata can be recorded by intensity readout or by additionalcoupling with an enzyme [20] fluorescent dye [24 25] orradioactive conjugates [26] Further developments of DNAbiosensors have exploited more powerful strategies includingquantitative real-time PCR [27ndash31] DNA microarrays [32]and surface plasmon resonance [33 34] These techniqueshave emerged as novel quantitative methods in microbiologybut are expensive and require trained laboratory technicians

A growing number of DNA biosensors have beendesigned for point-of-care diagnosis in the form of lateralflow (dipstick) nucleic acid test strips [19ndash24 26 36] Paper-based microfluidic bioassay appears to meet all the keystandards of a point-of-care diagnostic device [37] that isa sensitive and accurate assay that requires no complexequipment reagents or power sources and preferably amethod that is rapid small disposable and easy to use andtransport

This study aimed to develop a combination of PCR anddipstick-type DNA biosensors into one system as a rapidsensitive and visible multiplex analysis device to profilethe oral microbiota targeting the caries-associated bacte-ria Streptococcus mutans Streptococcus sobrinus Scardoviawiggsiae Actinomyces species and Veillonella parvula Thesebacteria have been known as crucial bacteria in the oralcavity as followsMutans streptococci in particular Smutansand S sobrinus have been thought to be associated withdental caries since they have frequently been isolated fromdental plaque biofilm where they produce large amountsof acids and extracellular polysaccharides which promotedental caries It has been reported that S wiggsiae has astrong association with early childhood caries in the USA[38] Actinomyces species are predominantly detected fromdental plaque biofilm periodotitis lesions and root surfacecaries [39] Veillonella species such as Veillonella parvulaare one of the indigenous bacteria of the oral cavity (dental

plaque biofilm and tongue coating) and the species obtaintheir energy by fermenting organic acids for example lactate[40] This metabolism has the potential to remove a potentdental-caries producing acid Most of the current DNAbiosensors utilize streptavidin-biotin interaction as the DNAcapture mechanism allowing only single detection of a targetdsDNA in one test To simultaneously test for several bacterialspecies different oligonucleotide strands were immobilizedin parallel on one strip In addition streptavidin-coatedcolored latex microspheres were used as reporters to givechromatic signals Semiquantitative information based onvisual intensity was attempted by a standardized 10-foldserial-dilution without any specialized reagents or instru-mentation

2 Materials and Methods

21 Bacterial Strains Five bacterial strains Streptococcusmutans NCTC 10449 Streptococcus sobrinus 6715 Scardoviawiggsiae F0424 Actinomyces oris WVU627 and Veillonellaparvula ATCC 10790 were cultured on CDC anaerobic 5sheep blood agar (BD Franklin Lakes NJ) plates and wereincubated at 37∘C for 3 days in an anaerobic glove box (ModelAZ-Hard Hirasawa Tokyo Japan) under an atmosphere of80 N

2 10 H

2 and 10 CO

2

22 Subjects and Sample Collection After obtaininginformed consent supragingival plaque (15mg) fromcaries-free enamel surfaces was sampled from 16 healthysubjects (mean age 318 plusmn 89 years range 23ndash54 years)The dentition (dental caries) status was recorded using theDMFT index (decayed missing and filled teeth) and thirdmolars were excluded No subjects had taken antibiotics for3 months prior to sampling Plaque samples were collectedusing sterile toothpicks and stored in 15mL sampling tubesat minus20∘C before analysis

23 DNA Extraction Genomic DNA was extracted fromdental plaque or 3-day-old bacterial cultures using the Insta-Gene Matrix Kit (Bio-Rad Laboratories Richmond CA)according to the manufacturerrsquos instructions

24 Polymerase Chain Reaction Direct PCR was performedusing the tagged species-specific primers listed in Table 1Each PCR mixture comprised 21 120583L of either plaque sam-ple DNA or bacterial genomic DNA 25120583L of Taq DNApolymerase (HotStar Taq PLUS Master Mix Qiagen GmbHHilden Germany) and 4 120583L of primer mixture PCR amplifi-cation was conducted in a PCRThermal Cycler MP (TaKaRaBiomedicals Ohtsu Shiga Japan) or an iCycler (Bio-RadLaboratories) programmed for 5min at 95∘C initial heatactivation and 30 cycles of 1min at 95∘C for denaturation1min at appropriate temperatures (in Table 1) for annealingand 15min at 72∘C for extension followed by 10min at 72∘Cfor final extension

25 Dipstick DNA Chromatography Dipstick DNA stripsand reagents were obtained commercially (TBA Co Sendai


Table1PC

Rprim

ersa

ndcond

ition

s

Bacterialstrain

Targeted

gene

Prim

ername

Sequ

ence

(51015840-31015840)

Size

(bp)

Ann

ealin

gtemperature

(∘ C)

Smutan

s16SrRNAgenes

Sm1(575F)

Tag1-X

-GGTC

AGGAAAG

TCTG

GAG

TAAAAG

GCT

A282

56Sm

2(856R)

Biotin-G

CGGTA

GCT

CCGGCA

CTAAG

CC

Ssobrinus

16SrRNAgenes

SobF

(54F

)Tag2-X-

CGGAC

TTGCT

CCAG

TGTT

ACTA

A546

56SobR

(599R)

Biotin-G

CCTT

TAAC

TTCA

GAC

TTAC

Swiggsiae

16SrRNAgenes

Scar

448F

Tag3-X-

GTG

GAC

TTTA

TCAAT

AAG

C172

51Scar

619R

Biotin-C

TACC

GTT

AAG

CAGTA

AG

Actin

omyces

species

16SrRNAgenes

AorF

Tag4-X-

GGCT

GCG

ATAC

CGTG

AGG

104

56Ao

rRBiotin-TCT

GCG

ATTA

CTAG

CGAC

TCC

Vparvula

rpoB

genes

Vp2696F

Tag5-X-

GAAG

CATT

GGAAG

CGAAAG

TTTC

G623

57Vp

3318R

Biotin-G

TGTA

ACAAG

GGAG

TACG

GAC

CAc

tinom


orisA

naeslu

ndiiandA

odontolyticus


C3rdquoTh

eSpacerC

3[th

ree-carbon

spacer(CH

2)3usingPh

osph

oram

ideC

3spacer

(GlenRe

searchSterling

VA)]isinserted

betweenTags

1(to

5)andar


er[35]


Absorptionpart

Control line






Marker (red line)

(a)


Biotin

Control line

Flow direction

Latex bead

Absorption part

TagX

V p

arvu

la

S m

utan

s

S so

brin

us

S w

iggs

iae

Test lines 1ndash5


Actin

omyc

essp

ecie

s

(b)



















PCR













100 pg 10 pg 1 pg 100 fg 10 fg 1 fg














Cr =DEr

(1)







nof

fiveg

iven

bacteriain


esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1PC

Rprim

ersa

ndcond

ition

s

Bacterialstrain

Targeted

gene

Prim

ername

Sequ

ence

(51015840-31015840)

Size

(bp)

Ann

ealin

gtemperature

(∘ C)

Smutan

s16SrRNAgenes

Sm1(575F)

Tag1-X

-GGTC

AGGAAAG

TCTG

GAG

TAAAAG

GCT

A282

56Sm

2(856R)

Biotin-G

CGGTA

GCT

CCGGCA

CTAAG

CC

Ssobrinus

16SrRNAgenes

SobF

(54F

)Tag2-X-

CGGAC

TTGCT

CCAG

TGTT

ACTA

A546

56SobR

(599R)

Biotin-G

CCTT

TAAC

TTCA

GAC

TTAC

Swiggsiae

16SrRNAgenes

Scar

448F

Tag3-X-

GTG

GAC

TTTA

TCAAT

AAG

C172

51Scar

619R

Biotin-C

TACC

GTT

AAG

CAGTA

AG

Actin

omyces

species

16SrRNAgenes

AorF

Tag4-X-

GGCT

GCG

ATAC

CGTG

AGG

104

56Ao

rRBiotin-TCT

GCG

ATTA

CTAG

CGAC

TCC

Vparvula

rpoB

genes

Vp2696F

Tag5-X-

GAAG

CATT

GGAAG

CGAAAG

TTTC

G623

57Vp

3318R

Biotin-G

TGTA

ACAAG

GGAG

TACG

GAC

CAc

tinom


orisA

naeslu

ndiiandA

odontolyticus


C3rdquoTh

eSpacerC

3[th

ree-carbon

spacer(CH

2)3usingPh

osph

oram

ideC

3spacer

(GlenRe

searchSterling

VA)]isinserted

betweenTags

1(to

5)andar


er[35]


Absorptionpart

Control line






Marker (red line)

(a)


Biotin

Control line

Flow direction

Latex bead

Absorption part

TagX

V p

arvu

la

S m

utan

s

S so

brin

us

S w

iggs

iae

Test lines 1ndash5


Actin

omyc

essp

ecie

s

(b)



















PCR













100 pg 10 pg 1 pg 100 fg 10 fg 1 fg














Cr =DEr

(1)







nof

fiveg

iven

bacteriain


esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Absorptionpart

Control line






Marker (red line)

(a)


Biotin

Control line

Flow direction

Latex bead

Absorption part

TagX

V p

arvu

la

S m

utan

s

S so

brin

us

S w

iggs

iae

Test lines 1ndash5


Actin

omyc

essp

ecie

s

(b)



















PCR













100 pg 10 pg 1 pg 100 fg 10 fg 1 fg














Cr =DEr

(1)







nof

fiveg

iven

bacteriain


esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















PCR













100 pg 10 pg 1 pg 100 fg 10 fg 1 fg














Cr =DEr

(1)







nof

fiveg

iven

bacteriain


esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



100 pg 10 pg 1 pg 100 fg 10 fg 1 fg














Cr =DEr

(1)







nof

fiveg

iven

bacteriain


esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



nof

fiveg

iven

bacteriain


esam

ples

Subject

Subject

Subject

12

34

56

78

Num

bero

fpositive

samples

()

910

11Num

bero

fpositive

samples

()

1213

1415

16Num

bero

fpositive

samples

()

Num

bero

fpositive

samplesfora

llsubjects(

)Lo

wDMFT

Mod

erateD

MFT

HighDMFT

00

00

01

22

34

48

1012

1212

Smutan

smdash

102

110

103

1010

102

7(875

)10

101

3(100)

110

102

102

105(100)

15(938)

Ssobrinus

mdashmdash

mdashmdash

mdashmdash

mdashmdash

0(00)

mdashmdash

mdash0(00)

mdashmdash

105

mdashmdash

1(200)

1(63)

Swiggsiae

mdashmdash

mdashmdash

105

mdashmdash

103

2(250)

mdashmdash

mdash0(00)

mdashmdash

103

104

mdash2(400)

4(250)

Actin

omyces

species

105

106

105

106

106

106

105

105

8(100)

106

106

105

3(100)

105

106

105

105

106

5(100)

16(100)

Vparvula

104

105

104

104

105

105

103

104

8(100)

mdash10

210

22(667)

105

102

104

104

102

5(100)

15(938)

Num

bero

fpo

sitiveb

acteria

lspecies

23

33

43

34

23

33

35

43



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusions




Acknowledgment


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article rapid and sensitive pcr-dipstick dna...

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