Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula

Real time PCR using TaqMan and SYBR Green for detection ofEnterobacter sakazakii in infant formulaYin Liu a ,Xiaoning Cai a ,Xia Zhang b ,Qili Gao b ,Xiaochuan Yang a ,Zejun Zheng a ,Maohuang Luo b ,Xitai Huang a ,*aDepartment of Biochemistry and Molecular Biology,College of Life Science,Nankai University,Tianjin 300071,ChinabTianjin Entry–Exit Inspection and Quarantine Bureau,Tianjin 300457,ChinaReceived 16March 2005;received in revised form 31May 2005;accepted 16June 2005Available online 3August 2005AbstractEnterobacter sakazakii is an emerging pathogen that causes meningitis,bacteremia,sepsis,and necrotizing enterocolitis in neonates and children.Powdered milk-based infant formulas have been associated with the E.sakazakii -related outbreaks in premature or other immunocompromised infants.In this study,we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI.The accuracy of two detections was tested by 35strains of E.sakazakii and 88non-E.sakazakii bacterial strains.The results showed that all of these E.sakazakii strains were positive reaction to the detections and all of the non-E.sakazakii strains were negative.The newly developed assays enable us to detect 1.1CFU/100g infant formula.And both of the assays can be accomplished within 2business pared to the traditional detection,the real time PCR procedures are quicker and simpler.In this study,we also developed a new method to design the primers,which can support multiple real time PCR with one pair of primers in SYBR Green detection.The detection methods are more sensitive and effective based on Two-Tm-Value of PCR.D 2005Elsevier B.V .All rights reserved.Keywords:Detection;E.sakazakii ;ITS;Real time PCR;SYBR Green;TaqMan1.IntroductionEnterobacter sakazakii is a serious infant patho-genic bacterium,which was firstly recognized as b yellow-pigmented Enterobacter cloacae Q in 1961(Urmenyi and Franklin,1961)in two cases of neo-nates.The majority of cases of E.sakazakii infectionin past reports were neonatal meningitis,sepsis and necrotizing enterocolitis and with 40–80%of the fatality rate to neonates (Simmons et al.,1989;Bier-ing et al.,1989;Burdette and Santos,2000;van Acker et al.,2001;Kandhai et al.,2004).Recently the cases of bacteremia and osteomyelitis in adults were reported too (Hawkins et al.,1991).Most reports suggested a role of powdered milk-based infant for-mulas as a vehicle for infection (Biering et al.,1989;Muytjens et al.,1988).Because a few cells of E.0167-7012/$-see front matter D 2005Elsevier B.V .All rights reserved.doi:10.1016/j.mimet.2005.06.007*Corresponding author.Tel./fax:+862223508874.E-mail address:huangxitai@ (X.Huang).Journal of Microbiological Methods 65(2006)21–31/locate/jmicmethsakazakii can cause severe impact on health in short time,detection of the pathogen has been an important subject in clinical research and food protection.In 2002,E.sakazakii had been ranked by The Interna-tional Commission for Microbiological Specification for Foods as a b Severe hazard for restricted popula-tions,life threatening or substantial chronic sequence or long duration Q bacteria(ICMSF,2002).In order to raise awareness of it,FAO/WHO requested a review be undertaken and two risk profiles of the organism had been published(Codex Alimentarius Commis-sion,2003a,b).The conventional methods for the isolation and identification of E.sakazakii from infant formula require enrichment culture(48h)and sub-culture to selective agar followed by phenotypic iden-tification;these procedures take up at least5days to obtain a result(FDA,2002).Real time PCR offers rapid,quantitative analysis for detection of food-borne pathogens(George et al.,2003;Andrew et al.,2003; Sylvie et al.,2004;Hajime et al.,2005;Zhu et al., 2005).The TaqMan system uses fluoregenic probes to detect PCR products by the exonuclease activity of Taq polymerase releases,a labeled reporter dye at the 5V end of the probe from the quencher dye at the3V end with each cycle of amplification.The SYBR Green fluorescent dye binds to the minor grooves of the amplified DNA during the primer annealing and extension steps of each PCR cycle.The16S rDNA and16S–23S rDNA internal tran-scribed spacer(ITS)sequence have been the most popular target for bacterial taxonomy.The bacterial rRNA operons are usually made up of16S rRNA gene,spacer,tRNA,23S rRNA gene,and5S rRNA gene.Many variations in lengths and sequences were found in ITS regions of different bacteria species. Accordingly,sequences of the bacterial ITS can be used for designing of genus or species-specific probes and PCR primers.The PCR methods based on ITS sequence have been used to detect Brucella spp., Mycobacteria,Streptococcus iniae,and Salmonella successively(Berridge et al.,1998;Condon et al., 1992,1995;Park et al.,2000;Rijpens et al.,1996; Tasi-Hsin et al.,2005).In the present study,ITS sequence was also employed to develop the real time PCR methods for detection of E.sakazakii with TaqMan and SYBR Green technology.We described the methods for rapid and sensitive detec-tion of E.sakazakii in infant formula by real time PCR using TaqMan and SYBR Green based on selec-tive enrichment by mLST and BHI.2.Materials and methods2.1.Bacterial strains,culture media and DNA extractionThirty-five E.sakazakii strains were used in this study.Three of them were collected from American Type Culture Collection(ATCC12868,ATCC29544, ATCC29004),and other32strains of E.sakazakii(15 from infant formula,17from other food)were col-lected from the culture collections of Tianjin Entry–Exit Inspection and Quarantine Bureau,Tianjin,P.R. China,(CIQ Tianjin).All of the isolated E.sakazakii were determined by the VITEK32system(bioMer-ieux,Co.,USA),and identified ultimately by the classical method reported by The Center for Food Safety and Applied Nutrition of U.S.Food and Drug Administration(FDA,2002).To test for the specificity of the real time PCR assay,88microorgan-isms(63Enterobacteriaceae strains and25strains of bacteria belonging to the other families)from the American Type Culture Collection(ATCC)(22 strains),China General Microbiological Culture Col-lection(CGMCC)(21strains),China Microbiological Culture Collection(B)[CMCC(B)](16strains),and CIQ Tianjin(Tianjin,P.R.China)(29strains)were tested.To extract genomic DNA of the bacteria,single colony was picked up from LB agar plate,then inocu-lated in3ml LB broth(10g bacto tryptone,5g yeast extract and5g NaCl in1l water)in a flask.The bacteria were cultured at378C for12–26h with shaking.To harvest the bacterial cells,1ml bacterial cultures(about108CFU/ml)were centrifuged at5000Âg for10min,the bacterial pastes were subjected to DNA extraction with a SK1201DNA extraction Kit (Sangon Biotech,China).The bacterial genomic DNA samples(in TE buffer)were stored atÀ208C for use.2.2.DNA sequencing of16S–23S rRNA internal transcribed spacer of E.sakazakiiTo determine the ITS sequences of the bacteria,a pair of universal primers were designed by alignmentY.Liu et al./Journal of Microbiological Methods65(2006)21–31 22of the3V terminal of the16S rRNA gene sequences and5V terminal sequences of the23S rRNA genes in GenBank.The b consensus Q sequences on both of the regions were chosen to be the universal primers for ITS amplification.The primers are UB-F(5V-GCTCG TGTNG TGANA TGTTG GGT-3V)in16S rRNA gene and UB-R(5V-GCGAT TTCYG AATGG GGRAA CCC-3V)in23S rRNA gene(N=A,T,C or G;Y=C or T;R=G or A).By using the universal primers,the ITS sequences of E.sakazakii were amplified and the PCR products were assayed by 1%agarose gel electrophoresis.The ITS DNA frag-ment of bands in gel(stained by EB)were cut out and the DNA were purified.Twelve of ITS sequences of E.sakazakii were determined in this study(sequenced by TaKaRa Biotech,China)and submitted to Gen-Bank.Their accession numbers are listed as follows: E.sakazakii ATCC12868,AY748352and AY748354;E.sakazakii ATCC29004,AY748353and AY748355;E.sakazakii ATCC29544,AY748356and AY748357;E.sakazakii GQL09,AY748360and AY748362;E. sakazakii GQL12,AY748358and AY748359; E. sakazakii GQL16,AY748361and AY748363.2.3.Real time PCR detection of E.sakazakii using TaqMan probeThe primers and probe were designed by alignment of the ITS containing tRNA Glu gene(G operon)of E. sakazakii.The forward primer:5V-CCGGAACAAG-CTGAAAATTGA-3V;the reverse primer:5V-TCTTC-GTGCTGCGAGTTTG-3V;and the fluoregenic probe for ITS sequence of E.sakazakii:5V-(FAM)ACTCT-GACACACCGCGCATTCCTG-3V(TAMRA)were assessed for species-specificity by a BLAST in NCBI homepage(search for homology to all of ITS sequences.The FAM(6-carboxy-fluorescein)was the fluorescein reporter.TAMRA is to be the quencher molecule to the oligonucleotide.To monitor the real time PCR of the DNA samples,an Internal Positive Control(IPC)was constructed by ligating the primers to an82-bp DNA fragment to create a123-bp recom-binant DNA that was subsequently cloned into pMD 18-T Vector(TaKaRa Code No.D504A).The recom-binant DNA was isolated from the transformed cells and was included in PCR reactions.The sequence of the TaqMan probe for the inserted DNA is:5V-(HEX) CCTTTGGCGTTTCCCGATGTCCGT-3V.HEX(5-hexachloro-6-carboxy-fluorescein)represents the other fluorescein reporter in different wavelength.TaqMan real time PCR reactions were performed in a25-A l reaction volume containing:12.5A l of Premix Ex Taq(2Â)buffer,1A l of the purified genomic DNA of bacteria,2A l of each of the10 A M oligonucleotide primers,1A l of3A M probe for E.sakazakii,1A l of3A M probe for IPC DNA,1A l of 103copies IPC DNA vector and an appropriate volume of water(all from TaKaRa Biotech,China). One well was used as non-template controls,which contained all reagents except the IPC and the DNA sample in every detection.The PCR were run on a Smart Cycler II System(Cepheid Co.,USA)using the following program:denaturation at958C for10s,45 cycles consisted of denaturation at958C for5s,62 8C for20s for annealing and extension.Fluorescence was quantified on-line and at the end point with the Smart Cycler Software(version1.6.3,Cepheid Co., USA)assigning TAMRA as the reference dye.Ct values were obtained based on a threshold pre-estab-lished at0.30.All PCR reactions were performed in triplicate.2.4.Real time PCR detection of E.sakazakii using SYBR GreenThe species-specific primers(forward primer:5V-TATAGGTTGTCTGCGAAAGCG-3V;reverse pri-mer:5V-GTCTTCGTGCTGCGAGTTTG-3V)were designed by alignment of the ITS sequence of E. sakazakii and the other bacteria in NCBI homepage. The forward primer was modified to amplify both of the ITS sequences containing tRNA Glu gene(G operon)and tRNA Ile+tRNA Ala genes(IA operon)in E.sakazakii(Fig.2)(see Section3.2.1).To monitor the quality of PCR,an IPC was constructed by ligat-ing the primers to a60-bp DNA fragment to create a 100-bp fragment that was subsequently cloned into pMD18-T Vector(TaKaRa Code No.D504A,Takara, China).The recombinant DNA isolated from the transformed cells was used to be IPC for the PCR reactions.The melt temperature(Tm)of amplified IPC segment from the recombinant plasmid is near 79.08C and different with the Tm of amplified seg-ments from ITS sequences of E.sakazakii.PCR was performed by using12.5A l SYBR Pre-mix Ex Taq(2Â)buffer,0.5A l of10A M of eachY.Liu et al./Journal of Microbiological Methods65(2006)21–3123primer,1A l of bacterial genomic DNA,1A l of103 copies IPC plasmid and an appropriate volume of water to make up25A l(TaKaRa Biotech,China). The PCR cycling parameters were as follows:initial denaturation at958C for10s,45cycles consisted of denaturation at958C for5s,628C for20s for annealing and extension.Following amplification,a melting curve analysis of the amplified DNA was performed at temperatures between54and958C, with the temperature increasing at a rate of0.28C/s. All PCR were performed in a LINE-GENE33 (BIOER Technology Co.,Japan).During the primer extension step of each amplification cycle,the increase in the fluorescence from the amplified DNA was recorded by using the SYBR Green optic channel set at a wavelength of495nm.The initial threshold value was set at30fluorescent units.2.5.Analysis of infant formula for E.sakazakii by real time PCRIn this study,100g infant formula were mixed with 900ml mLST(Tryptose20g,Lactose5g,K2HPO4 2.75g,KH2PO42.75g,NaCl34.22g,Sodium Laury Sulfate0.1g and Vancomycin10mg in1l of water)at 448C to dissolve completely.Following incubation at 448C for20h,500A l of culture was inoculated into5 ml BHI(37g BHI powder in1l water;Brain Heart Infusion;bioMerieux,Co.,USA)and incubated at37 8C.After another5-h incubation,1ml of the enrich-ment culture was collected and subjected to DNA extraction by SK1201DNA extraction kit.Concentra-tions of DNA were stored atÀ208C for real time PCR.2.6.Sensitivity of determinationsReal time PCR assays were evaluated for determin-ing sensitivity with E.sakazakii ATCC12868strain. For the determination,single colony was picked from a fresh culture and suspended in phosphate-buffered saline(pH7.0).Ten-fold serial dilutions,to10À9, were made and50A l of each dilution was spread in triplicate on LB agar.The plates were incubated for48 h at378C.The colonies on the plates were counted. For standard curves based on CFU,50A l was removed from the serial dilutions and inoculated into100g samples of infant formula which was confirmed to be culture negative of E.sakazakii by classical method of FDA(FDA,2002).The inoculated samples were incubated following the method of mLST–BHI accompanied enrichment as described in Section2.5.3.Result3.1.TaqMan real time PCR assay of E.sakazakii 3.1.1.Designation of primers and probeThe primers and probe of TaqMan real time PCR assay were selected from the ITS containing tRNA Glu gene(G operon)of E.sakazakii as described in Sec-tion2.3.There was no known non-E.sakazakii DNA sequences in the BLAST N databases being homo-logue to the primers and probe for E.sakazakii spe-cies.The primers can amplify a98-bp segment of the G operon of E.sakazakii.The position of the primers and probe were described as follows:the forward primer(369–389bp,DNA sequence accession num-ber AY748354in GenBank);the reverse primer(448–466bp);the probe(420–443bp).3.1.2.Specificity of primers and probeThirty-five strains of E.sakazakii were included in the test of specificity of the primers and probes.DNA was extracted from each strain and subjected to the TaqMan PCR.All of the DNA samples had a positive reaction in the assay.The specificity of the primers and probe was also tested against63Enterobacteria-ceae strains and25strains of bacteria belonging to other families.Most of them are food-borne organ-isms and pathogens.The DNA samples extracted from these strains did not show any fluorescent signal from FAM the TaqMan assay in34–35cycles(the HEX fluorescent signal from IPC detected about35 cycles).The results show that the primers and probes are specific to E.sakazakii and do not cause any amplification in all of the other relative bacteria.3.1.3.Sensitivity in pure bacterial cultureOne hundred microliters of genomic DNA sample of E.sakazakii were extracted from1ml of the bacterial culture which continued1.8Â108CFU of the bacter-ium,and10-fold dilutions of the DNA samples(1.8Â106–1.8Â101CFU/tube)were amplified with the Taq-Man assays in triplicate.The amplification curves forY.Liu et al./Journal of Microbiological Methods65(2006)21–31 24TaqMan assays showed that the result displayed a linear log correlative standard curve over the 10-fold dilution series (Fig.1A and B).The differences of the Cycle Threshold (Ct value)of the amplification curves between two 10-fold dilutions of DNA concentration is in a range of F 1.56cycles (Table 1).The results show that the detection limit of the TaqMan assay was 1.8Â101CFU approximately in 36.5cycles.3.1.4.Sensitivity in infant formulaInfant formula of five different brands (Table 2)in local supermarket was confirmed to be culture nega-tive for E.sakazakii by classical method of FDA(FDA,2002).One hundred grams sample of different brand (random selecting)was inoculated with 9of nine strains of E.sakazakii (the range of inoculated cell is 1.1Â100CFU to 3.9Â100CFU).The inocu-lated and three not-inoculated samples (Enfamil,S 26Gold,NAN 1,see Table 2)were incubated as the method described in Section 2.5.The DNA was extracted from culture for PCR assays.The 9inocu-lated samples were tested positive with Ct value of 16–18cycles (Table 2),while 3not-inoculated sam-ples were tested negative by this method (no FAM signal,but HEX signal with Ct value near 35cycles).According to the standard curve (Fig.1),the DNA sample in one tube was extracted from ~106CFU of the bacteria (18cycles).The detection limit for the ITS sequence TaqMan assay was 1.8Â101CFU at 36.5cycles,much less than 106CFU in BHI sample culture.Thus the sensitivity of the detection of patho-genic E.sakazakii in infant formula mLST–BHI enrichments by TaqMan real time PCR is 1.1CFU/100g infant formula by this procedure.In order to determine the dynamic range of the assay with infant formula,a dilution series of inocu-lants were tested.The 1-ml E.sakazakii ATCC 12868dilution series (2.9Â106–2.9Â101CFU/ml)were inoculated in 100g Enfamil infant formula (Mead Johnson,China).After mLST and BHI enrichment,DNA was extracted from 1ml BHI cultures.The7Fig.1.Sensitivity of TaqMan real time PCR detection of E.sakazakii of pure culture.(A)The amplifications of 10-fold serial dilution of E.sakazakii genomic DNA in TaqMan real time PCR.Sample number 1,genomic DNA dilution 1.8Â106CFU/tube;sample number 2,1.8Â105CFU;sample number 3,1.8Â104CFU;sample number 4,1.8Â103CFU;sample number 5,1.8Â102CFU;sample number 6,1.8Â101CFU.(B)Standard curves for E.sakazakii of TaqMan real time PCR.Standard curves were plotted for log cell number (CFU per tube)of the bacterium versus the number of cycles required to reach the Ct and based on the means of triplicate samples.The samples were derived from dilution of DNA extracted from the pure culture of E.sakazakii .The sample numbers correspond to the sample numbers shown in Table 1.Table 1Sensitivity of TaqMan and SYBR Green real time PCR for detection of E.sakazakii in pure culture a Sample number b Bacteria (CFU/tube)Ct value TaqMan SYBR Green 1 1.8Â10618.36F 1.3717.43F 0.232 1.8Â10522.25F 1.5621.29F 1.523 1.8Â10425.83F 0.825.39F 0.84 1.8Â10329.75F 0.9329.09F 0.775 1.8Â10233.5F 0.6231.98F 1.0361.8Â10136.51F 1.3132.86F 1.33aThe values are means F standard deviations for 3independent experiments.bThe sample numbers correspond to the lane numbers in Figs.1and 3.Y.Liu et al./Journal of Microbiological Methods 65(2006)21–3125inoculated samples were tested positive with Ct value of 15.11–21.03cycles (Table 3).The results suggested the range of TaqMan assay can be extended to a high CFU level (2.9Â106CFU).3.2.SYBR Green real time PCR detection of E.sakazakii3.2.1.Primers designIf there were two amplified DNA fragments,their melt temperature (Tm value)could be determined in SYBR Green PCR.Thus more information of detec-tion would be obtained.The internal transcribed spacer in E.sakazakii provides a proper target to take this advantage with a single pair of primers.There are two types of 16S–23S rDNA internal transcribed spacers in E.sakazakii .We designated the tDNA Glu containing operon as b G operon Q ,and tDNA Ile +tDNA Ala containing operon as b IA operon Q .We tried to use a single pair of primers to amplify and detect both of ITSs of G operon and IA operon in E.sakazakii .A species-specific DNA sequence was found in both of the ITS operons in E.sakazakii .The specific reverse primer was selected from this DNA sequence.The forward primer was chosen from the G operon,but with 4modified bases in order to complement to a region of IA operon.After optimizing specificity and amplification efficiency,the forward primer (21bp)could complement to G operon with 17bp and IA operon with 15bp (Fig.2).This pair of primers can support a multiple real time PCR for ITS amplification.A 285-bp segment in G operon and a 222-bp in IA operon were ampli-fied by the primers.The fluorescence signal in the PCR amplification was the same to that in the single PCR,but the curve of melt temperature showed two peaks of two amplification fragments (Fig.3A and B).The results were shown in all of the PCR reac-tions of 9strains E.sakazakii .And both the peaks show characteristic Tm of the 83.9F 1.08C for IA operon and 86.8F 0.98C for G operon (Table 2).The results suggested that this multiple PCR of SYBR Green is accurate and efficient for detection of E.sakazakii .3.2.2.Specificity of primers and probeThe primers of the SYBR Green assay were sub-jected to BLAST N database search to find any sequence similarities.There was no known non-E.sakazakii DNA sequences in the BLAST N databases with homology to the primers.Thirty-five strains of E.sakazakii were included in the tested,and DNA sam-ples extracted from these strains were amplified with the SYBR Green PCR.The specificity of the primers was also tested against 88strains of non-E.sakazakii bacteria in TaqMan assay.The genomic DNA (106Table 2Sensitivity of TaqMan and SYBR Green real time PCR for E.sakazakii detection in infant formula Inoculated strain Number of inoculated cells (CFU)Brand a of infant formulaCt value (cycles)Tm value in SYBR Green (8C)TaqMan SYBR Green IA operon G operon ATCC 12868 1.7Enfamil 18.1716.4383.386.1ATCC 29004 2.2S 26Gold 19.0415.7582.986.1ATCC 29544 1.8Wonder Sun 119.3016.5183.686.3M 30863 3.9Enfamil 17.7514.7583.986.9M 28612 1.9Enfamil 18.6117.2784.787.5GQL 01 1.7S 26Gold 17.8618.0083.686.8NKU08 2.7Wonder Sun 118.3215.5384.987.7NKU51 1.1NAN 118.6615.5583.086.0GQL121.8MORINAGA NL-3317.9816.1684.587.3aEnfamil (Mead Johnson,Guangzhou,China);S 26Gold (Wyeth,Shanghai,China);Wonder Sun 1(Wonder Sun,Heilongjiang,China);NAN 1,(Nestle,Denmark);MORINAGA NL-33(Morinaga,Japan).Table 3Dynamic range of the TaqMan real time PCR for E.sakazakii Number of inoculated cells Ct value 2.9Â10615.312.9Â10515.112.9Â10415.982.9Â10316.712.9Â10218.912.9Â10121.03Y.Liu et al./Journal of Microbiological Methods 65(2006)21–3126copies)of non-E.sakazakii cannot be amplified in the initial 35cycles in SYBR Green PCR.Furthermore,the DNA segments of amplification from the tested DNA also showed the distinct Tm value to that of E.sakazakii in SYBR Green PCR.3.2.3.Sensitivity in pure bacterial cultureDNA extracted from pure bacterial culture was amplified from the 10-fold dilution series (1.8Â106–1.8Â101CFU/tube)with the SYBR Green assays in triplicate.The difference of amplifi-cation curves in each DNA concentration is less than F 1.52cycles in Ct value (Table 1).Amplification curves for SYBR assays showed that the assay dis-played a linear log correlative standard curve up to 1.8Â102CFU/tube level (Fig.3C).Due to the inter-ference of the fluorescent signal from IPC,the Ct value of 1.8Â101CFU/tube was close to 1.8Â102CFU/tube.This interference can also be found in Tm value,the main peak was lower than the second peak and even the peak of IPC.And,the temperature of the peak was lower than the others (Fig.3B).Although the main peak from G operon signals were lower compared to sample without interference,they are still present.And,it was possible to distinguish true negative samples in the melting ing a single tube without IPC to detect the sample again is a more reliable assay,when the fluorescence signal of the sample appeared after 30cycles and the melting curve changed obviously.The Ct value of the 1.8Â101CFU/tube without IPC was 34.16F 1.29cycles,and the melting temperatures were 83.8F 0.28C for IA operon and 86.3F 0.38C for G operon.We found that the lowest level of detection of the ITS target sequence was 1.8Â101copies,and the expected melting temperatures were 83.9F 1.08C for IA operon and 86.8F 0.98C for G operon.3.2.4.Sensitivity in infant formulaA total of 9E.sakazakii strains in the enrichment samples were subjected to SYBR Green real time PCR (Table 2).All of them showed the positive fluorescence signal at a Ct value of 15to 18cycles and the expected melting temperature,approximately 83.9F 1.08C from IA operon and 86.8F 0.98C from G operon (Table 2).Because the SYBR Green PCR detection of E.sakazakii in infant formula was carried after the enrichment as the TaqMan detection,the sensitivity and the dynamic range of this assay is the same to that of the TaqMan detection (N 1.1Â100CFU /100g infant formula).4.DiscussionRecently,real time PCR has been shown to be useful in the detection of pathogenic bacteria in food products.The primer sets and probe sequences used in real time PCR provided an additional level of assay specificity that was demonstrated by using a variety of non-E.sakazakii species.None of the strains from these non-E.sakazakii bacterial species which have been tested generated a real time amplificationsignal.Fig.2.Primers’location of multiple real time PCR by single pair of primers in one PCR tube.Sequence 1,ITS of G operon in E.sakazakii ATCC 12868;the b forward Q ,forward primer region;the b reverse Q ,reverse primer region;sequence 2,ITS of IA operon in E.sakazakii ATCC 12868.Y.Liu et al./Journal of Microbiological Methods 65(2006)21–3127Nearly a single cell (1.1Â100CFU)in the real time PCR reaction after enrichment provided adequate tar-get for the detection within two business days.The dynamic range of detection (106to 101CFU in pure bacterial culture and 106to 100CFU in infant formula)demonstrates that the assay retains its sensitivity over a broad range of template DNA concentrations.Several conventional detections of this pathogen have been reported since 1988.A comparison of them to this real time PCR assay was shown in Table 4.Although the enrichment of mLST and BHI proce-dure was still required,the real time PCR was much more rapid than conventional methods.We have notattempted to demonstrate the sensitivity higher than 1CFU per collected samples,so the sensitivity is lower than the methods reported in some of references.As a target sequence of detection,multiple copies is another advantage of ITS.There are seven (three of IA operon and four of G operon)copies of ITS in a typical species of Enterobacteriaceae ,such as Escherichia coli ,Salmonella typhi ,Shigella flexneri .In this study,the ITS sequences of G operon are the target of TaqMan real time PCR.The sensitivity can be im-proved by a factor of 4than single copy of target gene.We found the interference from IPC reduced the sensitivity of the SYBR Green ing asingleFig.3.Sensitivity of SYBR Green real time PCR for detection of E.sakazakii of pure culture.(A)Amplification of ITS of E.sakazakii in SYBR Green real time PCR with serial dilutions of the genomic DNA.Fluorescence optic graph for the number of cycles versus the number of fluorescence units for each sample used to calculate the Ct value.Sample number 1,genomic DNA extracted from 1.8Â106CFU/tube;sample number 2,1.8Â105CFU;sample number 3,1.8Â104CFU;sample number 4,1.8Â103CFU;sample number 5,1.8Â102CFU;sample number 6,1.8Â101CFU.(B)Analysis of melting temperature on SYBR Green real time PCR of E.sakazakii genomic DNA.The results represented by a graph of number of derivative value [Àd (fluorescence units)/d (time)]temperature.Sample number 6,1.8Â101CFU show the interference from the IPC;Tm1,melting temperature of amplified DNA of IPC;Tm2,melting temperature of amplified DNA of IA operon ITS sequence;Tm3,melting temperature of amplified DNA of G operon ITS sequence.(C)Standard curves of SYBR Green real time PCR of genomic DNA from pure culture of E.sakazakii .The equation and the R 2include the interference of the data of 1.8Â101CFU.When the interference was eliminated,the equation should be y =À3.69x +43.486and R 2=0.9963.The sample numbers correspond to the sample numbers shown in Table 1.Y.Liu et al./Journal of Microbiological Methods 65(2006)21–3128。