Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

Development

of

a

propidium

monoazide-polymerase

chain

reaction

assay

for

detection

of

viable

Lactobacillus

brevis

in

beer

Yanlin

Ma

a,b

_,

_Yang

_Deng

a,b,∗

_,

_Zhenbo

_Xu

c

_,

_Junyan

_Liu

c

_,

_Jianjun

_Dong

a

_,

_Hua

_Yin

a

_,

Junhong

Yu

a

_,

_Zongming

_Chang

a

_,

_Dongfeng

_Wang

b,∗

a_{StateKeyLaboratoryofBiologicalFermentationEngineeringofBeer,TsingtaoBreweryCo.Ltd.,Qingdao,PRChina} b_{CollegeofFoodScienceandEngineering,OceanUniversityofChina,Qingdao,PRChina}

c_{CollegeofLightIndustryandFoodSciences,SouthChinaUniversityofTechnology,Guangzhou,PRChina}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26July2016 Accepted12November2016 Availableonline3June2017 AssociateEditor:AdalbertoPessoa

Keywords:

Propidiummonoazide Polymerasechainreaction Beerspoilagebacteria

Lactobacillusbrevis horA

a

b

s

t

r

a

c

t

Thespoilageofbeerbybacteriaisofgreatconcerntothebrewerasthiscanleadto tur-bidityandabnormalflavors.Thepolymerasechainreaction(PCR)methodfordetectionof beer-spoilagebacteriaishighlyspecificandprovidesresultsmuchfasterthantraditional microbiologytechniques.However, oneofthedrawbacksistheinabilitytodifferentiate betweenliveanddeadcells.Inthispaper,thecombinationofpropidiummonoazide(PMA) pretreatmentandconventionalPCRhadbeendescribed.TheestablishedPMA-PCR iden-tifiedbeerspoilageLactobacillusbrevisbasednotontheiridentity,butonthepresenceof

horAgenewhichweshowtobehighlycorrelatedwiththeabilityofbeerspoilageLABto growinbeer.Theresultssuggestedthattheuseof30␮g/mLorlessofPMAdidnotinhibit thePCRamplificationofDNAderivedfromviableL.breviscells.Theminimumamountof PMAtocompletelyinhibitthePCRamplificationofDNAderivedfromdeadL.breviscells was2.0␮g/mL.ThedetectionlimitofPMA-PCRassaydescribedherewasfoundtobe10 colonyformingunits(CFU)/reactionforthehorAgene.Moreover,thehorA-specificPMA-PCR assaysweresubjectedto18referenceisolates,representing100%specificitywithnofalse positiveamplificationobserved.OveralltheuseofhorA-specificPMA-PCRallowsfora sub-stantialreductioninthetimerequiredfordetectionofpotentialbeerspoilageL.brevisand efficientlydifferentiatesbetweenviableandnonviablecells.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthors.Tel.:+8653288891323.}

E-mails:dengyang719@hotmail.com(Y.Deng),wangdf@ouc.edu.cn(D.Wang).

http://dx.doi.org/10.1016/j.bjm.2016.11.012

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Limitedrangesofbacteriaarecapableofspoilingbeerowing tothepresenceofethanol(0.5–10%w/w),highcarbondioxide content(approximately0.5%w/v),relativelylowpH(3.8–4.7), extremelyreducedconcentrationofoxygen(<0.1ppm),lack ofnutritionandtheantibacterialeffectsofhopbitter com-pounds. Among the most problematic beer spoilers are severalspeciesofthegram-positive generalactobacilliand pediococci.1,2 _{Lactobacillusbrevisappearstobethemost} fre-quently isolated beer spoilage Lactobacillus species in beer and breweries. More than half of the bacterial incidents were caused bythis species.1 _{Itis oneofthe best-studied} beer spoilage bacteria and grows optimally at 30◦C and pH4–6.

Currentmethods ofdetectingbeer spoilagebacteria are time-consuming.Therefore,thebrewerrequiresarapid, accu-ratemethodasaqualitycontroltoolforscreeningsamples beforerelease into the marketplace. To shorten the detec-tiontime,severalmolecularmethodshavebeendevelopedfor thedetectionofbeerspoilage bacteriaparticularlyL.brevis,

basedontechniquessuchasthepolymerasechainreaction (PCR).3,4 _{However,one of the drawbacks is the inability to} discriminatebetweenliveanddeadcellsduetothe persis-tence of DNA after cell death. Ethidium monoazide (EMA) andpropidiummonoazide(PMA)were appliedpriortoPCR analysistocircumventthisproblem,allowingalive/dead dis-crimination ofbacteria.5–8 _{The intercalating dyecan enter} bacteriawithdamagedcellmembranesandcovalentlybind to genomic DNA upon exposure to light. Thebound DNA cannotbeamplified byPCR, thus preventingthe detection ofdeadcells.8_{AlthoughEMA/PMA-PCRhasbeenknownfor} several years, its applications in the brewing industry are scarce.

Beer spoilage L. brevis is generally resistant to hop compounds and thus can spoil beer.1,2 _{It is thought that}

L.brevisundergoesamulti-factorialhopadaptationprocess involving changes in metabolism and morphology, as well asthe moreenergy-dependent multidrugtransporter, hop-effluxmechanisms.9_{Theknownbeerspoilage-specificgenetic} markersforthesebacteriaarehitA,10_horA,11_andhorC,3,12_with

hitA and horC recentlybeingshown tobe less well associ-atedwithabilitytospoilbeer.4_{Anotherbeerspoilagerelated} gene,bsrA, was recentlyfoundtobe amarkerfor predict-ing beer spoilage ability ofPediococcus isolates.13 _Thewide andexclusivedistributionsofhorAinvariousbeerspoilageL. brevisisolatesindicatethepossibilityofspecies-independent detectionofbeerspoilageL.breviswiththegeneticmarker.4,14 Thehopresistancegene,horA,wasoriginallyidentifiedona 15.0kbplasmid,designatedaspRH45.2_{Thisplasmidwas} car-riedbyastrongbeerspoilageL.brevisstrainABBC45.pRH45 was initiallyrecognized asa plasmid, the copy number of whichmultipliedwiththehopadaptationofL.brevisABBC45.2 Consequently,the aimofthis study wasto investigatethe applicabilityofPMA-PCRtargetingthehorAgeneto discrim-inatebetweenviableand nonviableL.brevis nottoamplify otherbacteria.

Materials

and

methods

Bacterialstrains

Alist ofthe bacterialspeciestestedisprovidedinTable1, withthestrainscomprising13L.brevisand5non-lacticacid bacteria(5species).Allthesestrainsemployedinthisstudy wereisolatedandstoredinourlaboratorypreviously.15_Among them,thelacticacidbacteria(LAB)weregrownanaerobically indeManRogosaSharpe(MRS)broth(Oxoid,UK)at26◦Cfor 5days,whilethenon-LABwereincubatedat37◦Cand main-tainedinLuria-Bertani(LB)broth(Oxoid,UK)for24h.

Thebeerspoilage abilitywas investigatedusingthe tra-ditional “growth in beer test” described as Deng et al.16 Approximately102_cellsmL−1_{ofeachstrainwereinoculated} onto the apical surface of commercial bottled lager beers (filter-sterilized, 4.5% vol/volalcohol, pH 4.8, around 9 bit-ternessunits)understerileconditionsatroomtemperature. Bottle headspaces were flushedwith CO2 at aflow rateof 120mL/minforapproximately3mintoremovetheair.These bottleswerethentightlyrecappedwithmetallidsand incu-batedat26◦Candexaminedregularlyforvisiblegrowthfor upto1month.Bacteriacapableofgrowingineitherbeerwere consideredtobebeer-spoilers.Theabilityofthese18isolates togrowinbeerwasrecordedinTable1fordirectcomparison withtheresultsonpresenceorabsenceofhorAgene.

Inactivationofbacterialcells

Thebacteriawereheatedat65◦Cinawaterbathfor30min. Theresultingheat-treatedsampleswerecooledtoroom tem-peratureand theabsence ofviablecellsdetermined bythe passive dyeexclusionmethod16 _{usingaLive/DeadBacLight} bacterialviabilitykit(MolecularProbes,USA).Twofluorescent dyesSYTO9andpropidiumiodide(PI)were usedfollowing the manufacturer’s instructions toevaluatecell membrane integrityinthiskit.Cellsampleswerestainedwiththe mix-ture ofSYTO9 (5␮Mfinal concentration)and PI(30␮M)in 0.5Msodiumphosphate bufferatpH7.0,and incubatedin the darkatroomtemperaturefor20min.Thestained cells wereanalyzedundertheGuavaeasyCyte8HTflow cytome-ter(GuavaTechnologiesInc.,USA)usingbluelineexcitation at488nm.Resultsareexpressedasthenumberofviablecells permilliliterofthesamples.

DNA

isolation

and

PCR

assays

Genomic DNA were extracted from bacterial strains by using the TIANamp Bacteria DNA kit (Tiangen Biotech, China) according to the manufacturer’s instructions. The primer pairs specific to horA were designed as described

by Haakensen et al.4 _{The sequences of forward and}

reverse primers are 5-ATCCGGCGGTGGCAAATCA-3 and 5-AATCGCCAATCGTTGGCG-3 respectively, and amplify a 335-bpsegmentintheconservedregionofthehorAgene.15

Table1–Bacterialstrains,presenceofgenesandabilitytogrowinbeer.

Isolates Origins horAa _Growth(days)b

1:1c _{1:99 1:999 0:1}

Lacticacidbacteria

L.brevisCTT Brewery + + + − +(5) L.brevisDY Brewery + + + − +(6) L.brevisWCK Brewery + + + − +(3) L.brevisCN086 Brewery + + + − +(4) L.brevis2013-17 Brewery + + + − +(5) L.brevis86719 Brewery + + + − +(4) L.brevisCN3 Brewery + + + − +(6) L.brevisC598 Brewery + + + − +(3)

L.brevisC663 Pickledcabbage − − − − −

L.brevisCGMCC1.2028 Curedmeat − − − − −

L.brevisCGMCC1.3847 Milk − − − − −

L.brevisCGMCC1.1945 Wine − − − − −

L.brevisCGMCC1.2561 Wine − − − − −

Non-lacticacidbacteria

BacillussubtilisCGMCC1.3376 Soil − − − − −

StaphylococcusaureusCGMCC1.1809 Milkingmachine − − − − −

EnterococcusgallinarumCGMCC1.9125 Unknown − − − − −

EscherichiacoliO157:H7CGMCC1.2386 Humanfeces − − − − −

SalmonellaentericaCGMCC1.10603 Chicken − − − − −

a _{DeterminedbyhorA-specificPMA-PCR.}

b _{Thedetectiontimeisshowninparenthesis(days).}

c _{Differentmixturesofliveandheat-killedcellsin1:1,1:99,1:999,and0:1ratiosweresubjectedtohorA-specificPMA-PCRanalysis,respectively.}

+:visibleturbidityinbeer.

−:novisibleturbidityinbeer.

TaqDNApolymeraseandreactionmixturesweresuppliedasa kit(TaKaRaExTaq,TakaraBio,Japan).PCRreactionswere car-riedoutinaPTC-100Thermocycler(MJResearch,USA),and the particularcycling profilewere performedas previously described.4 _{Amplicons were detectedby electrophoresis in} 2.0%agarosegelscontainingethidiumbromide.Digitalimages wereobtainedusingaSpectrolineModelEAS-1000Electronic ArchivalSystem(SpectronicsCorp.,USA).NIHImage1.61 soft-warewasthenusedforrelativequantitationofDNAbands. Themeanvaluesofthefluorescenceintensitiesofbandswere derivedfromtriplicateindependentassays.TocheckforDNA amplificationfailureandtoconfirmthereliabilityofthePCR assay, PCR was performed witha synthetic DNA molecule nameinternalcontrol.Thenumberofcopyoftheinternal con-trolisverylow.Whentheconcentrationofthetargetsample ishigh,thecompetitionbetweenthe twoPCRcanconduct oftheinhibitionoftheinternalPCRproduct.Thus,aPCRis positivewhenwecandetectthetargetPCRproduct(horA);a PCRisnegativewhenwearenotabletodetectthetargetPCR productbutabletodetectthePCRinternalcontrolproduct. TheinternalcontrolDNAyieldeda706bpfragmentafterPCR amplification.Theprimersfortheinternalcontrolarea con-fidentialpartofthenowpurchasedkitfromTiangenBiotech, China.

DeterminationofthemaximumconcentrationofPMA thatdoesnotinhibitPCRfromL.brevisCTT

PMA(catalogueno.40013,Biotium Inc.,Hayward,CA, USA) wasdissolved in20% dimethylsulfoxide andadded tothe

suspensionsofL.brevisCTTatadensityof102_{colonyforming} units(CFU)/mLtofinalconcentrationsof1,5,10,20,30,50,75, and100␮g/mL,respectively.Sampleswerethenincubatedfor 10mininthedarkatroomtemperaturebeforebeingplaced inanicedcoolingbox.Subsequently,thetubeswereexposed for10minunlessotherwiseindicatedtoanLED(lightemitting diode)lightsource(470nm)atadistanceof15cmtoactivate andphotolysethePMA.Afterphoto-inducedcross-linkingof PMA,cellswerepelletedat10,000×gfor5minpriortoDNA extraction.

DeterminationoftheminimumconcentrationofPMA inhibitingPCRamplificationfromdeadL.brevis

AfterheattreatmentofL.brevisCTTculture(approximately 102_{CFU/mL)inmicrocentrifugetubes,theintercalatingdye} PMAwasaddedtocellsuspensionstofinalconcentrationsof 0.2,0.4,0.6,0.8,1.0,1.5,2.0,and3.0␮g/mL,respectively.The tubeswerethenplacedindarkfollowedbyagitationatroom temperaturefor10mintoallowthePMAtopenetratethe heat-treateddeadcells.TheywerefurtherexposedtotheLEDlight asdescribedaboveforviablecells.

Optimizationoflightexposuretimetoactiveand photolysePMA

CellsuspensionsofL.brevisCTTcontainingatotalof102_CFU in microcentrifuge tubes were treated withPMA ata final concentration of 3.0␮g/mL as described above followed by immediateremovaltothe darkfor10min.Thetubes were

thenplacedinanicedcoolingboxandsubsequentlyexposed tolightfor1,5,10,15,and20minrespectivelyatadistanceof 15cm.

SensitivityofhorA-specificPCRfordetectionofL.brevis CTT

ThedetectionlimitofPCRassaystargetingthehorAwas ascer-tainedbyminimalviablecellnumberofbacteria.About102 cellsmL−1ofL.brevisCTTwereinoculatedonto100-mLbottled beersasdescribedin“growthinbeertest”.Afterincubation at26◦C for10 days, thesuspensionsofL.brevis CTT were thendilutedwithbeerforserial10-fold,rangingfrom104_to 108 _{CFU/mL(1␮Lincluded inthePCRreaction). Anegative} controlwas performed using beer instead ofbacterial cul-ture.DNAisolationandPMA-PCRwereconductedasdescribed above.

SpecificityofPMA-PCRassaystargetingthehorAgene

Accordingtothepreviousresultsof“growthinbeertest”,the isolatescapableofgrowinginbeerweregrownanaerobically inlagerbeerat26◦Cfor10daysasdescribedabove,whereas othersweregrowninLBbrothat37◦Cfor24h.Theviableand heat-killedcellsofeachisolatepresentedinTable1werethen mixedin1:1,1:99,1:999,and0:1ratioscorrespondingto50%, 99%,99.9%,and100%nonviablecells,respectively.Thetotal numberofviableplusnonviablecellsinthe1mLvolumeswas keptconstantat105_{.Thelive/deadmixturesweretreatedwith} PMAatafinalconcentrationof3.0␮g/mLfor10mininthe dark,andthenexposedtothehalogenlampatadistanceof 15cmfor10minasdescribed above.Theresultingbacterial cellswerefurthersubjectedtoDNAextractionsandPMA-PCR analysisasaforementioned.

Statisticalanalysis

Student’sT-testwasusedtodeterminestatisticallysignificant differencesbetweenthemeanofthelogofgenomictargets derivedfromPCRandthemeanofthelogofCFUderivedfrom viabilityassayswiththeuseofSASsystemforwindows6.12 software.Aconfidenceintervalatthe99%level(p<0.01)was consideredinallcases.

Results

ThemaximumconcentrationofPMAnotinhibitingthePCR amplificationofDNAfromviablecells

When the culture of L. brevis CTT were treated with thePMA ataconcentrationof30␮g/mLorless,no signifi-cantinhibitionofamplification ofthe targetDNA occurred inthePMA-PCR procedure (Fig.1A).Statisticallysignificant (p<0.01) reductions in amplification of target DNA from viable cells occurred when the concentration of PMA was above 30␮g/mL. Increasing concentrations of PMA above 30␮g/mL resulted in proportional decreases in the ampli-ficationof target DNA from viablecells. Notable inhibition ofamplificationoccurredwhenthePMAconcentrationwas 100␮g/mL(Fig.1A).

1

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8000 6000 4000 2000 8000 6000 4000 2000 0 0 0.2 0.4 0.6 0.8 PMA concentration (mg/L) 1 1.5 2 3 0 0 Relativ e DNA intensity Relativ e DNA intensity 1 5 10 PMA concentration (mg/L) 20 30 50 75 100

A

B

Fig.1–(A)DeterminationofthemaximumamountofPMA

notinhibitingthePCRamplificationofDNAfromviableL.

brevisCTT.Lanes1–9,varyingconcentrationsofPMA(0,1,

5,10,20,30,50,75,and100mg/L,respectively).

(B)DeterminationoftheminimumamountofPMAto

inhibitthePCRamplificationofDNAfromheat-killedL.

brevis.Lanes1–9,varyingconcentrationsofPMA(0,0.2,0.4,

0.6,0.8,1.0,1.5,2.0,and3.0mg/L,respectively).

Microcentrifugetubescontainingatotalof102_viableor

nonviablecellsweretreatedwithdifferentconcentrations

ofPMAinthesetwotests,respectively.Top:typicalagarose

gelimageofPCRamplifiedproducts.Bottom:bargraphsof

fluorescenceintensityofcorrespondingDNAbandsderived

fromPCRwithrespecttocorrespondingconcentrationsof

PMA.

TheminimumconcentrationofPMArequiredtoinhibit theamplificationofDNAfromnonviablecells

TheamplificationoftargetDNAderivedfromheat-killedcells of L.brevis CTT was completely inhibited when such cells weresubjectedtotreatmentwithPMAataconcentrationof 2.0␮g/mLorhigher(Fig.1B).Incontrast,targetDNAfromthe nonviable cellswasamplifiedwhenthePMAconcentration was0.2to1.5␮g/mL.The2.0-␮g/mLconcentrationofPMAthat inhibitedtheamplificationofDNAfromtheheat-killedcellsis wellbelowthelevel(>30␮g/mL)thatresultedintheinhibition

CK

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5

CK

1

2

3

4

5

8000

A

B

6000 4000 2000 0 8000 6000 4000 2000 0 0 1 5 10 Light exposure time (min)

20 30 0 1 5 10

Light exposure time (min) 20 30

Relativ

e DNA band intensity

Relativ

e DNA band intensity

Fig.2–EffectsofthelightexposuretimetoachievecompletephotolysisoffreePMAinsuspensionsofliveandheat-killed

L.brevisCTT.Suspensions(1mL)containingatotalof102_{viableornonviablecellsweretreatedwithPMA(3.0␮g/mL).The}

tubeswerethenexposedtothehalogenlightfor1to20minatadistanceof15cm.(A)Top,typicalagarosegelimageofPCR

amplifiedproductsderivedfromDNAofviablecells.LaneCK:viablecellswithoutPMAtreatmentasacontrol,lanes1–5,

exposuretimeforphotolysisoffreePMA(1,5,10,15,and20min,respectively).(B)Thesameprotocolaswith(A)except

withdeadcells.

ofamplificationofDNAderivedfromviablecells.PMAata con-centrationof3.0␮g/mLshouldbethereforeideallysuitablefor discriminationofDNAfromamixedpopulationofviableand nonviablebeerspoilageL.brevisCTTbythePMA-PCR.

Theoptimallightexposuretimeaftertreatmentoflive anddeadcellswithPMA

ThesuspensionsofL.brevisCTTatadensityof1×105_CFU/mL wereexposedto3.0␮g/mLPMAasdescribedabove.Thefree PMAinthesuspensionsofL.breviscellswerethenexposedto thehalogenlampfor1,5,10,15,and20min,respectively. Inac-tivationoffreePMAwasachievedwithlightexposurefrom 1minto20min(Fig.2A),whichwasreflectedintheabsence ofadecreaseinDNAamplificationwitheachoftheselight exposuretimescomparedtotheamplificationofDNAfrom thecontrolcellsnottreatedwithPMA(p<0.01).Lightexposure ofdeadcellssimilarlytreatedwithPMAfor1minorlonger completelypreventedamplification(Fig.2B).Lightexposure for1minorlongerwasthereforefoundtobesatisfactoryfor selectiveamplification oftarget DNA from the viablecells. Finally,alightexposureperiodof10minwaschosenforour subsequentstandardizedassaytoensureinactivationoffree PMAwhichiscapableofbindingtoDNAfollowingcelllysis andtherebypreventingamplificationoftargetDNAfromthe viablecells.

SensitivityofhorA-specificPCRfordetectionofL.brevis CTT

ThesensitivityofhorA-specificPCRassayswasascertainedby minimalviablecellnumberofL.brevisCTT.Ascanbeseen inFig.3,thedetection limitofPCRassayswasfoundtobe 10CFU/reaction(PCRwaspositiveforsamplecontaining104 CFU/mL,with1␮Lincludedinthereactionsystem)forthe

horAgene. CK 5 4 3 2 1 8000 6000 4000 2000 0 0 CFU/PCR 105 104 103 102 101 Relativ

e DNA band intensity

Fig.3–SensitivityofPMA-PCRassaystargetingthehorA

genefordetectionofL.brevisCTT.Thedetectionlimitwas

evaluatedwithseriallydilutedcellssuspensions

containingbetween104_and108_{viableL.breviscells}

(namely101_to105_{CFU/PCR).Thenumberofviablecellsis}

increaseddecimallyfromline1(105_{CFU/PCR)throughlane}

5(101_{CFU/PCR).LaneCK:beerasanegativecontrol.}

Differentiationbetweenliveanddeadcellsinmixed samplesbyPMA-PCR

TheestablishedhorA-specificPCRassayswereappliedfor dif-ferentiation betweenviableand nonviablecells ofthe beer spoilageL.brevisandnon-spoilagebacterialistedinTable1. WhentheDNAfromaconstantnumberoftotalbacteriawas 1×105_mL−1_{derivedfromdifferentratiosofviableand} nonvi-ablecells,thePCRamplificationoftargetDNAfromthedead cellswaseffectivelyinhibitedby3.0␮g/mLofPMAasexpected (datanotshown).Eightofthesestrainsweredetectedtobe

positiveforhorAbyPMA-PCRasshowninTable1.The fluores-cencewasnotinfluencedbythepresenceoftheDNAfromthe deadcells,evenwhentheheat-killedcellsconstituted99.9%of thetotalcellpopulation.Thisobservationisinaccordwiththe resultsofgrowthinbeer.Allthe8horA-positiveisolateswere capableofgrowinginbeertested,showingthehorA-specific

PMA-PCR hadavery high specificityfordetectingthe beer spoilageL.brevis(Table1).

Discussion

Inourwork,theobservationthat100%ofhorAPCR-positive LABisolatescould growinbeerreinforcesthefactthatthe

horA PCR accurately detected L. brevis capable of rapidly causing beer spoilage. The horA gene has homology to adenosinetriphosphate (ATP)-bindingtransportertoexport

trans-isohumulone,preventingitsaccumulationinthe intra-cellularspace.17_{ThegenehorAisconsideredasasignificant} andeffectivepredictorofbeerspoilagecapability.9

Sami et al. initially described a PCR assay designed to amplifytheATP-bindingcassette(ABC)regionofhorA.11But thehorAPCRprimersdescribedbySamietal.arenotoptimal, and a low PCR cycle number must be used to avoid non-specificamplificationofnon-horA,ABC-containinggenes.In 2006,Suzukietal.attemptedtoimproveonthehorAprimers ofSamiet al.9 _{ThehorA-specific PCRmethoddevelopedby} Suzukietal.allowsmorespecificandsensitivedetermination ofthebeer-spoilageabilityofL.brevis.9_{Subsequently,} Haak-ensenetal.designedamultiplexPCRtodetectfourputative beer-spoilage-associated genes (horA,hitA, horC,and ORF5) thatincludedthe16SrRNAgeneasaninternalcontrol.4_Their datafurtherindicatedthatassayingforthepresenceofhorA

washighlyaccurateinpredictingthebeer-spoilagepotential ofL.brevisisolates.4_{However,someinactivatedcellsmaybe} detectedwiththeseabovehorA-specificPCRmethodsasno differentiationismadebetweenDNAfromviableor nonvi-ablecells.Thisisaconsiderableweaknessoftheconventional PCRmethodologyasitcanleadtofalse-positiveresults.18,19 Apromisingstrategytoavoidthisissuereliesontheuseof nucleicacidintercalatingdyes,suchasPMAorEMA,asa sam-plepretreatmentpriortoPCR.

PMAis able toenter cells with compromised cell walls andintercalateintoDNAofdeadcells.Onlightexposurea covalentDNA-PMAcomplexisformed,however,thisbound DNA cannot be PCR amplified. Here we have found that PMA treatment prior to PCR generally reduces the signal fromdeadcells.Inthisstudy,thedetectionlimitwasfound to be 10 CFU/PCR reaction which corroborated the earlier findings.9_{EvaluationoftheefficacyofPMA-PCRandEMA-PCR} onmixturesofviableanddeadcellsthusdemonstratedthat PMA is effective in selectively allowing PCR amplification from viablecells when inthe presence ofDNA from dead cells.5,6 _{Incontrast,EMAwasshown tosignificantlyinhibit} PCRamplificationfromviablecells,aswellasfromdeadcells. Although an intact cell membrane should be an effective barriertoEMAbecauseofthechargeofthemolecule,various reportshavealsosuggestthatEMAmaypenetrateviablecells ofsomebacterialspecies.6,8,20_{ThefirstreportofEMA} inhib-itingPCRamplification from viablecells wasmade forthe

bacterium Anoxybacillus.21 _{Nocker et al.}6 _subsequently reportedthatalthoughEMAandPMAwereequallyefficientin preventingPCRamplificationfromdeadStaphylococcusaureus, Listeriamonocytogenes,Micrococcusluteus,Mycobacteriumavium

and Streptococcus sobrinuscells, EMAalsocaused inhibition of PCR amplification from viable cells of these species. These studies indicate that EMA cannot be considered to be membrane impermeable for all bacterial species. PMA appearstohavetheadvantageoverEMAofnotpenetrating membranesofviablecells,yeteffectivelyenteringdamaged ordeadcells.5_{TheenhancedselectivityofPMAismostlikely} duetothehigherchargeofthePMAmolecule(PMAhastwo positivecharges, whileEMAhasonlyone),and thegreater impermeabilitythroughtheintactcellmembrane.6,22

InspiteofthedemonstratedefficiencyofthePMA-PCRfor the preventionofamplification fromdead L.breviscells in pureculture,theapplicationofthismethodtorealfood matri-cessuchasbreweryproductsinthebeerproductionprocess stillrequiresfurtherevaluation.Itwouldalsobeofinterest to apply the developed PMA-PCR on other species ofbeer spoilagebacteriainordertoensurethatthemethodis applica-bleasanentity.Additionally,theassaysdescribedinthiswork allowqualitativeviable/deaddifferentiationinpurecultures ofL.brevis.Forfutureapplications,however,thehorA-specific

PMA-PCRmethodinthisstudyshouldbeadaptedfor quanti-tativeviable/deaddifferentiationinmixedbacterialcultures byusingquantitativereal-timePCR.Real-timePCRisthemost widelyappliedtechnologyfordirectquantificationofcellsin mixedsamples.Real-timePCRisincreasinglybeingusedfor directdetectionandquantificationofpathogensinfoodsand environmental orclinical samples.23–25 In2007, Haakensen etal.describedaquantitativereal-timePCRassaywithoutthe pretreatmentofPMA/EMAforthespecificdetectionofhorA

gene.26 _{Thisisthefirstreportonthequantitativedetection} ofbeer-spoilage LAB.ThehorAreal-time PCRdevelopedby Haakensenetal.wasprovedtobeaneffectivetoolforrapid, accuratedetectionandquantitationofL.brevisinbeer.26

Inconclusion,thetreatmentofsamplescontainingbeer spoilageL.breviswithPMApriortoPCRhasgreatpotential for reducing the false-positivesignal from nonviable cells. By specificallytargetingorganismscapableofbeer spoilage throughcombineduseofPMAandhorA-specificPCR, brew-eryqualitycontrollaboratorieswillbeabletomakerapidand accuratepredictionsregardingthepotentialbeerspoilage out-comeofcontaminationbyL.brevis.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwasfinanciallysupportedbyChinaPostdoctoral Sci-enceFoundationfundedproject(no.2015M582063),theOpen ResearchFundofStateKeyLaboratoryofBiological Fermenta-tionEngineeringofBeer(no.K2016001)andtheOpenProject ProgramofStateKeyLaboratoryofFoodScienceand Technol-ogy,NanchangUniversity(no.SKLF-KF-201415).

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