rapid and direct quantitative detection of viable bifidobacteria in probiotic yogurt by combination...

Rapid and direct quantitative detection of viable bifidobacteriain probiotic yogurt by combination of ethidium monoazide andreal-time PCR using a molecular beacon approach

XC Meng*, R Pang, C Wang and LQ Wang

Key Laboratory of Dairy Science (Ministry of Education), Northeast Agricultural University, Harbin, People’s Republic of China

Received 11 September 2009; accepted for publication 13 July 2010; first published online 8 September 2010

The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beaconprobe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated inthis work. A real-time PCR with molecular beacon assay was developed to determine genusBifidobacterium quantitatively in order to increase the sensitivity and specificity of assay. EMAwas used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allowdetection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific for Bifid.was designed. The specificity of the probes ensures that no signal is generated by non-targetamplicons. Linear regression analysis demonstrated a good correlation (R2=0.9948) between theEMA real-time PCR results and the plate counting, and real-time quantitative PCR resultscorrelated adequately with enumeration of bifidobacteria by culture for commercial probioticyogurt. This culture-independent approach is promising for the direct and rapid detection ofviable bifidobacteria in commercial probiotic yogurt, and the detection can be carried outwithin 4 h. The detection limit for this method is about 104 cell/ml. In conclusion, the directquantitative EMA real-time PCR assay based on molecular beacon described in this research is arapid and quantitative method.

Keywords: Bifidobacterium, real-time PCR, molecular beacon, ethidium monoazide.

Bifidobacteria are common members of human gastroin-testinal microflora and are known to contribute to healthbenefits to humans (Ventura et al. 2007). Because theyplay an important role in the control of a balanced intes-tinal microflora, bifidobaceria are considered as probioticand are used in preparation of functional foods as ingre-dients. From a functional food perspective, the presence oflive bifidobacteria in products is particularly indispens-able. As a general recommendation and in the absence of‘‘dose response’’ data for many strains, it is suggested thatprobiotic products should contain at least 107 CFU/g (ml)(Ishibashi & Shimura, 1993). Probiotic yogurt is preferablecarrier of bifidobacteria. We know it is easy to enumeratethe amount of bifidobacteria in products by the platecounting when only bifidobacterial strains are added toproducts. However bifidobacterial strains are commonlyused together with other probiotic strains or other lacticacid bacteria strains.

Culture-based methods, especially selective media arevery useful in enumeration of bifidobacteria. It has pre-viously been shown that a number of selective media,such as LP-MRS (Vinderola & Reinheimer, 1999), NPNL-MRS agar (Martin & Chou, 1992), AMC (Payne et al. 1999)and OG-MRS (Lim et al. 1995) were suggested to performa selective enumeration of bifidobacteria in various milkproducts. However these culture-based methods are labourintensive and time consuming. Triggered by these short-comings, culture-independent methods such as real-timePCR (Masco et al. 2007) and enzymatic-colorimetric assays(Bibiloni et al. 2000) have been developed. In recentyears, several molecular methods have been described: aPCR on the hsp60 gene for a rapid detection of bifido-bacteria in raw milk and raw milk cheese samples(Delcenserie et al. 2005); a PCR-denaturing gradient gelelectrophoresis (DGGE) technique for the screening of themicrobial composition of probiotic products (includingbifidobacteria) (Fasoli et al. 2003); the quantitative real-time PCR for quantification of Bifid. in human fecal samples(Gueimonde et al. 2004), in probiotic products (Mascoet al. 2007) and in bacterial mixtures (Vitali et al. 2003);*For correspondence; e-mail : [email protected]

Journal of Dairy Research (2010) 77 498–504. f Proprietors of Journal of Dairy Research 2010 498doi:10.1017/S0022029910000658

the pulsed-field gel electrophoresis (PFGE) method foridentification of bifidobacterial strains in probiotic milkproducts (Grand et al. 2003). Of all the advanced methodsbased on PCR techniques and newly-developed methodsbased on signal or probe enlargement, real-time PCR tech-nology has been attracting attention as a reliable method(Vitali et al. 2003). However, conventional real-time PCRmethods do not distinguish viable cells from dead cells.

The selective detection of viable but not dead bacteriais a major issue in nucleic acid-based detection (Rudi et al.2002). Ethidium monoazide (EMA) is a DNA intercalatingagent (Bolton & Kearns, 1978) which is reported to selec-tively penetrate the membranes of dead cells, but is pur-portedly unable to penetrate the intact membranes ofviable cells (Rudi et al. 2002; Nogva et al. 2003). Nogvaet al. (2003) reported that dead cells stained by EMA didnot result in DNA amplification by real-time PCR. Rudi et al.(2002), Nogva et al. (2003) and Wang et al. (2009) usedthe EMA to discriminate between living and dead cells ofEscherichia coli, Salmonella sp., Listeria monocytogenesand Campylobacter jejuni by PCR. However, it has beenreported that DNA amplification from viable cells of cer-tain bacteria, such as Enterobacter sakazakii (Cawthorn &Witthuhn, 2008), List. monocytogenes (Nocker et al.2006; Flekna et al. 2007; Pan & Breidt, 2007) and Camp.jejuni (Flekna et al. 2007), can also be negatively influ-enced by EMA staining.

In the present work, the potential of EMA real-time PCRmethod for rapid detection of viable bifidobacteria presentin probiotic yogurt was evaluated. For this purpose, a real-time PCR with molecular beacon assay was developed todetermine Bifid. quantitatively in order to increase thesensitivity and specificity of assay. EMA treatment proce-dures were optimized for selectively amplifying a DNAtarget sequence via molecular beacon and real-time PCRfrom only viable cells of Bifid. in the presence of deadcells in probiotic yogurt. Furthermore, the EMA real-timePCR method was applied to enumerate viable bifido-bacteria in commercial probiotic yogurt.

Materials and Methods

Bacterial strains, media and culture conditions

The strains used in this study are shown in Table 1. A totalof 28 bacterial strains, including bifidobacterial (n=14)and non-bifidobacterial (n=14) strains, were used toevaluate the specificity of the molecular beacon probe andprimers. Bifidobacterial strains were cultivated overnightin trypticase peptone yeast (TPY) broth under anaerobicconditions (80% N2, 10% H2, 10% CO2) at 37 8C, andother lactic acid bacterial strains were cultivated overnightin Man Rogosa Sharpe (MRS) (Oxoid) broth under aerobicconditions at 37 8C. Esch. coli was cultured aerobically innutrition agar at 37 8C. All cells were harvested from theearly exponential growth phase. Colony forming units(CFU) of bifidobacterial cultures were determined by serial

dilutions of cultures in maximum recovery diluents (MRD)(Oxoid) followed by pour plating with TPY. Plates wereincubated at 37 8C for 3 d under anaerobic conditions.

Preparation of probiotic yogurt samples containingbifidobacteria

The bifidobacterial cultures were prepared separately, andthen added into the yogurt, in order to obtain high countsof bifidobacteria and avoid microbial competition with the

Table 1. The bacterial strains used for determination of thespecificity of the real-time PCR based on molecular beaconprobes in this study

Species Strain Source

Bifidobacterium longum ATCC 15707 CGMCC†Bifidobacterium longum KLDS 2.0505 KLDS-DICC‡Bifidobacterium adolescentis KLDS 2.0003 KLDS-DICCBifidobacterium breve KLDS 2.0004 KLDS-DICCBifidobacterium adolescentis KLDS 2.0005 KLDS-DICCBifidobacterium adolescentis KLDS 2.0506 KLDS-DICCBifidobacterium bifidum KLDS 2.0507 KLDS-DICCBifidobacterium bifidum KLDS 2.0006 KLDS-DICCBifidobacterium breve KLDS 2.0007 KLDS-DICCBifidobacterium bifidum KLDS 2.9502 KLDS-DICCBifidobacterium bifidum KLDS 2.0502 KLDS-DICCBifidobacterium bifidum KLDS 2.0504 KLDS-DICCBifidobacterium infantis KLDS 2.0503 KLDS-DICCBifidobacterium lactis KLDS 2.0501 KLDS-DICCLactobacillus delbrueckii subsp. KLDS 1.0204 KLDS-DICCbulgaricus KLDS 1.0207 KLDS-DICCLactobacillus delbrueckii subsp. KLDS 1.0208 KLDS-DICCbulgaricus KLDS 1.0209 KLDS-DICCLactobacillus delbrueckii subsp. KLDS 1.0210 KLDS-DICCbulgaricus KLDS 3.0201 KLDS-DICCLactobacillus delbrueckii subsp. KLDS 3.0202 KLDS-DICCbulgaricus KLDS 3.0203 KLDS-DICCLactobacillus delbrueckii subsp. KLDS 3.0205 KLDS-DICCbulgaricus KLDS 4.0316 KLDS-DICCStreptococcus salivarius subsp. KLDS 4.0309 KLDS-DICCthermophilus KLDS 1.0327 KLDS-DICCStreptococcus salivarius subsp. KLDS 1.0319 KLDS-DICCthermophilus ATCC 25922 RIAMAS·Streptococcus salivarius subsp.thermophilusStreptococcus salivarius subsp.thermophilusLactococcus lactisLactococcus lactisLactobacillus acidophilusLactobacillus caseiEscherichia coli

ATCC: American Type Culture Collection; KLDS: Key Laboratory of Dairy

Science (Education Ministry of China)

† China General Microbiological Culture Collection Center (CGMCC)

‡Dairy Industiral Microbiology Culture Collection Center (KLDS) (KLDS-

DICC)

·Research Institute of Applied Microbiology, Heilongjiang of Academy of

Science

Rapid detection of viable bifidobacteria in yogurt 499

starter cultures. Bifid. longum ATCC 15707 cultures weremade by incubation overnight at 37 8C with a 1% (v/v)inoculum in TPY under anaerobic conditions. Cells ofculture were harvested by centrifugation at 2,500rg for10 min and resuspended in 10% (w/v) sterilized recon-stituted skim milk (RSM), the cell density of bifidobacteriain RSM was adjusted to 1011 CFU/ml approximately.

Full-fat milk powder (Nestle) was reconstituted withwater at 13% (w/v) total solids, and 7% (w/w) sucrosewas added, then the total was pasteurized at 90 8C for15 min. After pasteurization, the milk mixture was cooleddown to 45 8C and commercial yogurt starter culturescontained Streptococcus salivarius subsp. thermophilusand Lactobacillus delbrueckii subsp. bulgaricus (YF-L902;Chr. Hansen, Denmark) was added. The milk mixturewas stirred gently, and was brought to incubate at 43 8Ctill pH 4.6 was obtained. This fermented milk is defined asyogurt samples. RSM suspension containing bifidobacteria(1011 CFU/ml) was added to above yogurt with a 1% (v/v)inoculum, and the probiotic yogurt samples containingbifidobacteria were obtained. Both yogurt samples andprobiotic yogurt samples were taken and kept at 4 8Covernight.

Total DNA extraction from probiotic yogurt samples

Total DNA of probiotic yogurt samples were extracted aspreviously described by Rademaker et al. (2006) withmodifications. Probiotic yogurt samples of 25 ml were di-luted in 225 ml of sterile PBS (phosphate-buffered saline,composed of 137 mM-NaCl, 2.7 mM-KCl, 10 mM-Na2HPO4,2.0 mM-KH2PO4, pH 7.4). One milliliter of 10-fold dilutionprobiotic yogurt samples were mixed with 100 ml 18% (w/v) sodium citrate and 50 ml 1 M-NaOH at room temperature,then the mixture were centrifuged at 6600rg for 10 minin the Eppendorff tubes. The pelletwaswashed in PBS buffer.The washed pellet was resuspended in 100 ml MilliQ wa-ter, and was mixed with 100 ml 2% (w/v) Triton X-100(Sigma). This mixture was heated at 100 8C for 10 min,then immediately cooled in ice-water, and centrifuged at6600rg for 10 min and the supernatant was stored at–20 8C until its use.

Primers and molecular beacon probes design

The sequences of the genus-specific primers for genusBifid. were designed on the basis of 16S rRNA and/or 16SrDNA sequences which were downloaded from theGenBank database (http://www.ncbi.nlm.nih.gov). The mul-tiple alignments were prepared by the program Clustal W(http://www.ebi.ac.uk). The genus-specific primers for Bifid.were designed by primer design software (Primer 5.0)after comparison of unique bifidobacterial primer se-quences with a large number of reference primer se-quences (Kaufmann et al. 1997; Venema et al. 2003;Vitali et al. 2003; Matsuki et al. 2004). Secondary struc-tures and dimers formation were controlled by the Oligo

Analyzer 6.0 Software (http://www.bioon.com/Soft/Class1/Index.html). The sequences of the primers were as follows,Bif-F : 5k-TCTGGCTCMGGATGAACGC-3k ; Bif-R: 5k-CACC-GTTACACCGGGAATTC-3k. These primers have a lengthof about 20–25 bases, a G/C content of over 50%, and a Tmof about 58 8C. Primers used in this study were synthesizedby TaKaRa (TaKaRa Biotechnology [Dalian] Co. Ltd.).Primers were resuspended in TE buffer (1 mM-EDTA-10 mM-Tris-HCl buffer, pH 8.0) and stored at –20 8C. Ali-quots (10 mM) of each primer were prepared and used forPCR.

Comparative analysis of the primary nucleotide se-quences of the 16S rRNA of Bifid. longum ATCC 15707(accession number M58739) amplicon generated by con-ventional PCR was used for the design of a molecularbeacon probe. Fluorescein (6-FAM) was chosen as thechromophore (excitation at 488 nm and mission at 519 nm)and DABCYL was used as the quencher. Fluorescein andDABCYL were coupled to the 5’ and 3’ ends of the beaconprobe respectively. The sequence of the probe (occupyingposition 161–180 of the 16S rDNA of Bifid. longum ATCC15707) with fluorophore and quencher moieties was:5k-FAM-CCAGGCATCCGGCATTACCACCCGTCCTGG-3k-DABCYL. Molecular beacon probes used in this studywere synthesized by Sangon Company (Shanghai, China).

Molecular beacon real-time PCR

Real-time PCR amplification reactions were performedwith the 7500 Real Time PCR System (Applied Biosystems,USA) using above primers and molecular beacon probes.Thermal cycling conditions were specified as follows: 1cycle of 94 8C for 3 min; 40 cycles of 94 8C for 30 s, 40 8Cfor 35 s, 59 8C for 30 s and 72 8C for 1 min. Fluorescentmeasurements were recorded during each first annealingstep (40 8C for 35 s). Data were analyzed using theSequence Detection System software (version 1.9.1)(Applied Biosystems). For each PCR, 5 ml of template DNAwas added to 45 ml PCR master (5 ml of 10rPCR buffer[TIANWEI, Beijing, China], 2.5 mM-MgCl2, 0.8 mM-eachprimer, 0.4 mM-beacons, 200 mM-each of the four deoxy-nucleoside triphosphates [TIANWEI, Beijing, China], 1.25Uof Taq DNA polymerase [TIANWEI, Beijing, China], and16.5 ml water). The PCR buffer contained ROX as the ref-erence dye for normalization of the reactions. Any possiblefluctuations in ROX signal were used to correct the samplesignal. Non-template controls were included in all PCRruns and tested negative.

Genomic DNA of bifidobacterial culture was extractedas previously described by Matsuki et al. (2004), andgenomic DNA of non-bifidobacterial culture was extractedby using TIANamp Bacteria DNA Kit (TIANGEN BIOTECH[Beijing] Co., LTD). The genomic DNA of 14 bifido-bacterial strains and 14 non-bifidobacterial strains wasamplified to assess the ability of the real-time PCR assay todiscriminate Bifid. from other bacterial strains as describedabove. All detections were performed in triplicate.

500 XC Meng and others

Influence of EMA concentration on differentiationbetween viable and heat-killed Bifid. longumATCC 15707 in probiotic yogurt samples containingbifidobacteria

Ethidium monoazide bromide (Sigma) was dissolved indimethylformamide (DMF) (Sigma) to yield a stock EMAsolution containing 1 mg/ml, which was stored at –20 8Cin the dark. This EMA stock solution of different volumes(0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 5 ml) were added re-spectively into 6 microcentrifuge tubes containing 1.0 ml of10-fold dilution probiotic yogurt samples with 109 CFU/mlheat-treated dead bifidobacteria at 100 8C for 5 min and6 microcentrifuge tubes containing 1.0 ml of 10-fold di-lution probiotic yogurt samples with 109 CFU/ml viablebifidobacteria. These sample tubes were then placed in thedark at room temperature for 5 min, and subsequentlyplaced on ice and exposed to light for 2 min at a distanceof 20 cm from the light source (OSRAM with a 650-W

halogen light bulb, Osram, Germany) to activate andphotolyse the EMA. The EMA was handled as a carcino-gen. The DNA was extracted from the EMA-treated sam-ples, and real-time PCR was performed to detect theamplification reactions of DNA as described above. Allreal-time PCR assays were performed at least three inde-pendent times with the mean Ct values and standard de-viations reported.

Standard curve

The standard curve used to make correlation betweenviable counts determined by real-time PCR coupled withEMA and plate counts using the selective agar media wasobtained from viable cells of Bifid. longum ATCC 15707 inyogurt. Bifid. longum ATCC 15707 culture was seriallydiluted with sterilized water to obtain a range of CFUequivalent to approximately 2r108 CFU/ml, 2r107 CFU/ml, 2r106 CFU/ml, 2r105 CFU/ml, 2r104 CFU/ml, andthen centrifuged at 2, 500rg for 10 min respect-ively. These bacterial cell pellets were resuspended in theyogurt which contained viable Strep. salivarius subsp.

Table 2. Representative real-time PCR amplification results obtained from 14 bifidobacterial strains and 14 non-bifidobacterial strains

Species Ct±SD Species Ct±SD

Bifidobacterium longum ATCC 15707 16.32±0.03 Lactobacillus delbrueckii subsp. bulgaricus KLDS 1.0204 —Bifidobacterium longum KLDS 2.0505 14.31±0.21 Lactobacillus delbrueckii subsp. bulgaricus KLDS 1.0207 —Bifidobacterium adolescentis KLDS 2.0003 19.55±0.12 Lactobacillus delbrueckii subsp. bulgaricus KLDS 1.0208 —Bifidobacterium breve KLDS 2.0004 15.85±0.13 Lactobacillus delbrueckii subsp. bulgaricus KLDS 1.0209 —Bifidobacterium adolescentis KLDS 2.0005 19.20±0.08 Lactobacillus delbrueckii subsp. bulgaricus KLDS 1.0210 —Bifidobacterium adolescentis KLDS 2.0506 22.08±0.33 Streptococcus salivarius subsp. thermophilus KLDS 3.0201 —Bifidobacterium bifidum KLDS 2.0507 16.43±0.25 Streptococcus salivarius subsp. thermophilus KLDS 3.0202 —Bifidobacterium bifidum KLDS 2.0006 16.61±0.39 Streptococcus salivarius subsp. thermophilus KLDS 3.0203 —Bifidobacterium breve KLDS 2.0007 15.72±0.12 Streptococcus salivarius subsp. thermophilus KLDS 3.0205 —Bifidobacterium bifidum KLDS 2.9502 15.12±0.53 Lactobacillus lactis KLDS 4.0316 —Bifidobacterium bifidum KLDS 2.0502 16.73±0.44 Lactobacillus lactis KLDS 4.0309 —Bifidobacterium bifidum KLDS 2.0504 14.63±0.15 Lactobacillus acidophilus KLDS 1.0327 —Bifidobacterium infantis KLDS 2.0503 14.75±0.35 Lactobacillus casei KLDS 1.0319 —Bifidobacterium lactis KLDS 2.0501 16.29±0.21 Escherichia coli ATCC 25922 —

The values are means of triplicates±standard deviation (SD)

‘‘—’’ means no fluorescence signal and no Ct value

Table 3. Real-time PCR amplification results of DNA derivedfrom dead and viable cells of Bifidobacterium in yogurt treatedwith EMA of different concentrations

Dead cells Viable cells

Concentrationof EMA mg/ml Ct±SD

Concentrationof EMA mg/ml Ct±SD

0 23.84±0.08 0 23.17±0.130.5 25.39±0.38 0.5 23.24±0.161.0 26.87±0.25 1.0 23.47±0.361.5 — 1.5 23.55±0.102.0 — 2.0 24.84±0.172.5 — 2.5 26.53±0.355.0 — 5.0 26.85±0.09

The values are means of triplicates±standard deviation (SD)

‘‘—’’ means no fluorescence signal and no Ct value

y = -3.2933x + 41.044

R2 = 0.9948

05

10152025303540

3 4 5 6 7 8 9

LogCO

Ct

Fig. 1. Standard curve for quantifying viable Bifidobacterium inprobiotic yogurt containing bifidobacteria by real-time PCRcombined with ethidium monoazide based on the molecularbeacon probe.


thermophilus and Lb. delbrueckii subsp.bulgaricus. EMA treatment of probiotic yogurt and DNAisolation were carried out as described above, thenamplified by molecular beacon probe real-time PCR. Bifid.longum ATCC 15707 counts in different suspension weredetermined by LP-MRS agar (Vinderola & Reinheimer,1999). By plotting the log CFU/ml against the Ct value, alinear relationship was formed.

Comparison of the EMA molecular beacon real-timePCR assay and plate counts for quantitative detectionof bifidobacteria in commercial probiotic yogurt

Probiotic yogurts were purchased from Carrefour super-market (Harbin, China). The total bifidobacteria of probioticyogurts were detected by real-time PCR, and the viablebifidobacteria of probiotic yogurts were detected by EMAreal-time PCR method established in this study and platecounting respectively. The results obtainedby thesemethodswere compared to elucidate the accuracy of EMA real-timePCR method. LP-MRS agar was used for enumerating bifi-dobacteria in probiotic yogurt (Vinderola & Reinheimer,1999). All detections were performed in triplicate.

Statistical analysis

All cell amounts obtained by EMA real-time PCR and CFUcounts were converted to log counts prior to statisticalanalysis. Paired Student’s tests were performed usingMicrosoft Excel software (Windows XP) and SAS statisticssoftware 8.0.

Results

Specificity of the real-time PCR based on molecularbeacon probe

Genomic DNA extracted from bifidobacterial (n=14) andnon-bifidobacterial strains (n=14) was amplified to assess

the ability of the real-time PCR assay to discriminate bifi-dobacteria from non-bifidobacteria. Real-time PCR studiesshowed that all of the bifidobacterial strains were positivelydetected, and the non-bifidobacterial strains were not de-tected by the real-time PCR assay (Table 2).

Influence of EMA concentration on differentiationbetween viable and heat-killed Bifid. longum ATCC15707 in probiotic yogurt containing bifidobacteria

The amplification of DNA derived from dead cells stillexisted when EMA concentration was less than 1.5 mg/ml,otherwise there was no detectable difference in thePCR signals from dead bifidobacteria when EMA concen-trations was 1.5 mg/ml or higher (Table 3). It was showedthat the amplification of target DNA derived from deadcells of bifidobacteria in the real-time PCR was completelyinhibited when EMA concentrations were 1.5 mg/mlor higher.

When the EMA concentration was 2.0 mg/ml or less,little or no inhibition of the amplification of the targetDNA derived from viable cells occurred in the real-timePCR procedure (Table 3). However there was significantinhibition of amplification when the EMA concentrationwas 2.5 or 5.0 mg/ml (P<0.05), but it also had the Ct values(Table 3). The Ct values obtained from EMA treatment(1.5 mg/ml) for viable bifidobacteria were not significantlydifferent (P>0.05) from the Ct value of the control.Therefore, it was concluded that the minimum concen-tration of EMA was 1.5 mg/ml by this method.

Standard curve

A linear relation (y= –3.2933x+41.044, x representativelog CFU/ml, y representative Ct values, R2=0.9948)between bacterial concentrations and Ct values was ob-tained in the range of 104–108 CFU/ml for probiotic yogurtcontaining bifidobacteria using real-time PCR based on themolecular beacon probe (Fig. 1).

Table 4. The detection results of total and viable bifidobacteria in commercial probiotic yogurt determined by real-time PCR, EMAreal-time PCR assay and plate counting (n=3)

Numberof yogurt

Total bifidobacteriadetermined by real-time PCR

Viable bifidobacteriadetermined by EMA real-time PCR Viable bifidobacteria

determined by platecounting log cfu/ml±SDCt±SD log cells/ml±SD Ct±SD log cells/ml±SD

1 21.60±1.14 5.91±0.35a 24.25±1.03 5.09±0.31b 5.12±0.05b

2 23.48±0.99 5.33±0.30a 25.94±1.01 4.59±0.31b 4.72±0.13b

3 23.43±1.09 5.35±0.33a 25.08±0.74 4.75±0.22b 4.74±0.13b

4 21.32±1.12 5.99±0.34a 23.56±0.29 5.31±0.09b 5.52±0.24b

5 17.50±1.72 7.15±0.52a 22.87±2.27 5.51±0.69b 5.59±0.32b

6 21.03±1.77 6.07±0.54a 23.45±1.18 5.34±0.36a 5.29±0.07a

7 23.26±0.24 5.40±0.07a 24.68±0.95 4.87±0.29b 4.84±0.06b

8 23.02±0.38 6.01±0.65a 23.03±0.38 5.47±0.11a 5.62±0.33a

9 15.02±0.93 7.9±0.28a 20.11±1.04 6.16±0.32b 6.17±0.12b

a,b mean in row with a common superscript do not differ (P>0.05)


Comparison of the EMA molecular beacon real-timePCR assay and plate counting for quantitative detectionof bifidobacteria in commercial probiotic yogurt

Comparison of the viable bifidobacterial cells obtained byculture and EMA real-time PCR showed that there wasgood correlation (Table 4). The number of viable bifido-bacteria in probiotic yogurts determined by EMA real-timePCR was somewhat lower than that detected by platecounting, however there was no significant difference be-tween them (P>0.05). The number of total bifidobacteriafor some samples determined by real-time PCR was higherthan that of the viable bifidobacteria determined by EMAreal-time PCR and plate counting significantly (P<0.05).Table 4 illustrated EMA real-time PCR assay established inthis investigation was a good method for direct determi-nation of viable bifidobacteria in probiotic yogurts, and thedetection can be carried out within 4 h.

Discussion

Binding dyes and hybridization probes are all used to de-tect DNA amplification in real-time PCR experiments. Forthe former, the primers have to be highly specific to avoidsynthesis of undesirable PCR products. The later uses a setof primers for amplification of a DNA fragment, and inaddition, a highly specific probe labelled with a fluorogenicsubstance that is able to pair with the nascent DNA fragmentemitting a signal upon interaction. The overall specificityof the PCR reaction is thus substantially enhanced. A real-time quantitative PCR method, based on the use of probeslabelled with a stable fluorescent lanthanide chelate, isdeveloped for the quantification of different human faecalbifidobacterial populations (Gueimonde et al. 2007).Furthermore, molecular beacons, due to their stable stem-and-loop structure, have been demonstrated to besignificantly more specific than dyes such as SYBR green Iand other types of probes (Gubala & Proll, 2006).

In the present study, we designed the primer set of Bif-F/Bif-R which is genus-specific for Bifid. The real-time PCRmethod presented here is highly specific. The specificity ofthe primers and probes was tested both by identity searchesin a nucleotide database (GenBank) and by the screeningof 14 representative bifidobacterial and 14 non-bifido-bacterial strains. The specificity of the probes ensures thatno signal is generated by non-target amplicons. An im-portant aspect of a microbiological detection assay is theability to detect only viable bacteria in the samples andcultivation techniques have been relied on as a measure ofcell viability. EMA treatment was selected because it hasbeen shown that EMA can prevent amplification of DNAfrom dead bacteria in the PCR assay (Nogva et al. 2003;Wang et al. 2009).

Varying EMA concentrations contribute a significantdifference in Ct values for either viable or killed cellssamples. EMA with final concentration of 100 mg/ml areused to treat Esch. coli O157:H7, Sal. enterica serovar

Typhimurium cultures (Nocker & Camper, 2006) andCamp. jejuni (Rudi et al. 2005a). But the optimum finalconcentration of EMA was 1.5 mg/ml when probiotic yogurtcontaining bifidobacteria was treated by EMA in thisresearch.

Comparison of this direct quantitative method withstandard culture technique for bifidobacterial counts inprobiotic yogurt revealed that the assay described herein hasthe potential to be applied on a routine basis to probioticyogurt for the detection of bifidobacteria. In agreementwith previous publications introducing the application ofEMA in combination with quantitative PCR to differentiatebetween viable and dead bacterial cells (Nogva et al. 2003;Rudi et al. 2005b), the efficacy of this DNA-intercalatingdye in selecting against DNA from dead cells was con-firmed (Nocker & Camper, 2006).

Of course, recent studies found that propidium mono-azide (PMA) may exert less influence on DNA amplificationfrom certain viable cells than EMA. PMA has been usedto detect viable Ent. sakazakii, Esch. coli O157:H7, List.monocytogenes and Camp. jejuni with success (Nockeret al. 2006; Flekna et al. 2007; Pan & Breidt, 2007;Cawthorn & Witthuhn, 2008). Continuation of this workusing PMA instead of EMA would be an interesting nextstep.

In conclusion, the quantitative EMA molecular beacon-based real-time PCR assay described in this researchare reliable and rapid methods for the quantification ofviable bifidobacteria in probiotic yogurt. This culture-independent approach is promising for the direct and rapiddetection of viable bifidobacteria in probiotic yogurt.Using this protocol, it was possible to detect bifidobacteriain probiotic yogurt within 4 h. The detection limit forquantitative real-time PCR analysis of bifidobacteria isabout 104 cell/ml (g). The protocol has the potential for usein monitoring level of viable bifidobacteria in probioticyogurt during manufacture or storage. This method is fasterbut requires expensive reagents and real-time PCR system.

This research was supported by grant ZJN03-3 from theHeilongjiang Province Natural Sciences Fund, China and National863 Program (2008AA10Z335) of People’s Republic of China.

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