# Comprehensive and Rapid Real-Time PCR Analysis of  21 Foodborne Outbreaks

Authors: Hiroshi Fukushima; Kazunori Katsube; Yoshie Tsunomori; Ryoko Kishi; Junko Atsuta; Yuko Akiba
Journal: International Journal of Microbiology
 (2009)

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## Abstract

A set of four duplex SYBR Green I PCR (SG-PCR) assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within  2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.


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## Body

## 1. Introduction

The introduction of real-time PCR in foodborne outbreak investigations provides
an opportunity  for rapid detection of pathogens in food and clinical settings [1].
The benefits to public health administration from rapid real-time PCR assays
are most notable after comprehensive and rapid detection of bacteria. The results can
quickly inform a public health administrator about the causative pathogens of foodborne
outbreak, allowing a more accurate, effective, and timely response. Abubakar et
al. [2] implied in the Health Technology Assessment program (now part of the
National Institute for Health Research, UK) that the feasibility of conversion
to rapid methods such as multiplex PCR and DNA microarrays is dependent on
localized considerations, including the community prevalence rates for specific
pathogens, the skill base, and subsequent training costs for laboratory staff
and spare capacity available to ensure adequate laboratory space for new
equipment. Although these tests look promising, further studies are necessary
to assess their usefulness [2].Apart from saving
time, real-time PCR is sensitive, highly specific and offers the potential for
quantification [3]. The risk of cross-contamination is significantly reduced,
and high-throughput performance and automation are possible since no post-PCR
manipulations are required [4]. In principle, two different chemistries are
available for real-time detection of PCR products: fluorescent probes that bind
specifically to certain DNA sequences and fluorescent dyes that intercalate into
any double-stranded DNA. Fluorescent-probe based real-time PCR (TaqMan PCR)
studies to detect causative pathogens from foodborne outbreaks in feces using
TaqMan probes have been carried out [3–6]. TaqMan PCR assays require the availability of
primers and probes that must be selected according to very rigid criteria. Use of simple, cheaper double-stranded DNA-binding dye SYBR green I for
detection of PCR amplicons (SG-PCR) overcomes this limitation. Therefore, real-time PCR could be applied without the need for fluorescent probes [7].
In the absence of probes, the specificityof the reaction is determined on
the basis of the melting temperature (Tm).
The advantages of SG-PCR over TaqMan PCR
include the relative simplicity and reduced cost of SYBR Green I compared to TaqMan probes [8]. Recently,
the application of SG-PCR for the detection of foodborne bacteria in different
samples has been increased [1, 9–12]. Duplex SG-PCR assays have been carried out to detect causative bacteria in feces from foodborne outbreaks [4, 10, 13].We previously reported a set of four duplex SG-PCR assays for one-step
differentiation of 8 genes of foodborne pathogens in DNA extracted from 5 feces
using 32 capillary tubes of LightCycler (Roche). The first run was for the
detection of 8 main foodborne pathogens and the second run was for the other
pathogens. We reported here that improved diagnostic duplex SG-PCR assays were
upgraded with new highly sensitive primer pairs for 11 foodborne pathogens. These
assays successfully identified the causative pathogens of foodborne outbreaks
caused by enteropathogenicEscherichia
coli, enterohemorrhagic E. coli, astA-positive E. coli, Plesiomonas shigelloides, Vibrio parahaemolyticus,
Campylobacter jejuni, Clostridium
perfringens,
Bacillus cereus, or Staphylococcus aureus in 21 cases from 2002 to 2007. This assay is
simple, rapid, inexpensive, reliable as well as suitable for comprehensive, rapid detection of causative pathogens in foodborne
outbreaks.


## 2. Material and Methods

### 2.1. Bacterial Strains

The 27 foodborne bacteria used in this
study areE. coli (enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)), Shigellasonnei, Salmonella Enteritidis, Yersinia
enterocolitica, Yersinia pseudotuberculosis, Providencia alcalifaciens, Plesiomonas
shigelloides, Campylobacter jejuni, C. coli, Vibrio cholerae, TDH-positive V.
parahaemolyticus, TRH-positive V. parahaemolyticus, Aeromonas hydrophila, Staphylococcus aureus, emetic Bacillus cereus, enterotoxigenic B. cereus, and Clostridium perfringens (Table 1). Bacterial cultures and viable-cell counting were
described in a previous report [10]. For template DNA of each foodborne
pathogen as a PCR control, 200 μL of each bacterial culture (108 CFU/mL) was treated with a QIAamp DNA Stool Mini kit (Qiagen) in the same
procedure as the following stool treatments.Table 1
Bacterial strains assayed by SYBR Green I PCR
Bacterial strainsSourcesePCR results with each primer set (see Table2)eaeJMS1JMS2LTSTaEAST-1aggR-ZvirAStyinvayadA-XPAGPSGABCC ceuEhlyAtdhtrhAHH1FemBces-TMSGGAPEscherichia coli -EPECO55(eaeA)EC-2736b+−−−−−−−−−−−−−−−−−−−−−E. coli -EPEC
O153 (eaeA and astA)EC-2649b+−−−−+−−−−−−−−−−−−−−−−E. coli -EHEC O26:H11(Stx1)SE-02005++−−−−−−−−−−−−−−−−−−−−E. coli -EHEC O157:H7 (Stx2)SE020025+−+−−−−−−−−−−−−−−−−−−−E. coli -EHEC O157:H7
(Stx1 and Stx2)SE-02027+++−−−−−−−−−−−−−−−−−−−E. coli -ETEC O148
(LT, ST and astA)EC-3515b−−−+++−−−−−−−−−−−−−−−−E. coli -ETEC O169
(ST and astA)EC-4725b−−−−++−−−−−−−−−−−−−−−−E. coli- EAEC O111 (aggR and astA)EC-4131b−−−−−++−−−−−−−−−−−−−−−E. coli -EIEC O124:HNM (virA)EA32a−−−−−−−+−−−−−−−−−−−−−−Shigella sonneiI00031−−−−−−−+−−−−−−−−−−−−−−Salmonella EnteritidisSal-2339−−−−−−−−+−−−−−−−−−−−−−Yersinia enterocolitica O3/B4Pa241−−−−−−−−−+−−−−−−−−−−−−Y. pseudotuberculosis O4bSP988−−−−−−−−−+−−−−−−−−−−−−Providencia alcalifaciensNIID124C−−−−−−−−−−+−−−−−−−−−−−Plesiomonas shigelloidesNIID123C−−−−−−−−−−−+−−−−−−−−−−Campylobacter jejuniSC 009−−−−−−−−−−−−+−−−−−−−−−Campylobacter coliSC 011−−−−−−−−−−−−−+−−−−−−−−Vibrio cholerae O1ATCC14035−−−−−−−−−−−−−−+−−−−−−−V. cholerae O139NIID63-93C−−−−−−−−−−−−−−+−−−−−−−V. cholerae non- O1SVP84−−−−−−−−−−−−−−+−−−−−−−V. parahaemoliticus O3:K6 (tdh)SVP02−−−−−−−−−−−−−−−+−−−−−−V. parahaemoliticus O3:K6 (trh)NIIDK4C−−−−−−−−−−−−−−−−+−−−−−Aeromonas hydrophila O1ATCC7966−−−−−−−−−−−−−−−−−+−−−−Staphylococcus aureusSS 05e−−−−−−−−−−−−−−−−−−+−−−EmeticBacillus cereusNo.127e−−−−−−−−−−−−−−−−−−−++−EnterotoxigenicB. cereusNo.1e−−−−−−−−−−−−−−−−−−−−+−Clostridium perfringensH2d−−−−−−−−−−−−−−−−−−−−−+Strain kindly donated by K. Sugiyamaa,
Shizuoka Prefectural Institute of Public Health, Shizuoka; J. Yatsuyanagib, Akita
Prefectural Institute of Public Health, Akita; M. Tamura and E. Arakawac,
Ofther strains except for ATCC numbers are our own
collections.


### 2.2. Primer Design

The
22 primer pairs used in this study for the detection ofE. coli (EIEC, EPEC, EHEC, ETEC, and EAEC), Salmonellaenterica, Shigella spp., Y. enterocolitica, Y. pseudotuberculosis, P. alcalifaciens, C. jejuni,
C. coli, V. cholerae,  V. parahaemolyticus, A. hydrophila, P. shigelloides, S. aureus, C. perfringens,
and B. cereus
were
described in our previous reports [10, 13] for cases 1 to 19. The newly designed 22
primer pairs listed in Table 2 were used for cases 19 to 21. In this study, 10
primer pairs (marked with * in Table 2) were newly designed or selected from
earlier publications (see Table 2 references). The 4 primer pairs (ces, yadA-X,
CCceuE, and aggR-Z) were newly designed. The ces primer was constructed from cereulide synthetase gene of emetic B. cereus [4], the
yadA-X primer from yadA gene on the
plasmid present in virulent Yersinia spp. [24], the CCceuE primer from ceuE gene
encoding of a lipoprotein component of a binding-protein-dependent transport
system for the siderophore enterochelin of C.
coli [25], and the aggR-Z primer from aggR gene encoding of a transcriptional activator
for EAEC aggregative adherence fimbria I expression [26]. To
determine the specific primers ces,
yadA-X, CCceuE, and aggR-Z, the genes of ces, yadA, ceuE, and aggR that were expected to be unique were selected with the Basic
Local Alignment Search Tool (BLAST) program within GenBank and were designed by Biosearch Technologies Inc. (USA). Other primer pairs were
those used in earlier publications (see Table 2 references). All
oligonucleotide primers were synthesized by Invitrogen (Yokohama, Japan)
or Biosearch Technologies Inc. (USA).Table 2
22 pairs of specific primers for SYBR Green I PCR
Primer set for duplex PCRSpecies and subgroupsTarget genePCR primersGenBank accession no.locationProduct size (bp)Tm valuesaReferencesNameForward or reversprimers' sequences (5' - 3')First run1Escherichia
colieaeA*beaeF2CATTGATCAGGATTTTTCTGGTGATAZ11541899-92410683.2±0.2[14]EPEC and EHECRCTCATGCGGAAATAGCCGTTA1000-979Staphylococcus
aureusfemB*FemBfwAATTAACGAAATGGGCAGAAACAAF106850277-2999380.8±0.3[15]rvTGCGCAACACCCTGAACTT370-3512Campylobacter
jejuniC. jejuni- specific DNA*ABFCTGAATTTGATACCTTAAGTGCAGCAL1111683811358679.1±0.4[8]DNARAGGCACGCCTAAACCTATAGCT381185EAECastAEAST-1SGCCATCAACACAGTATATCCL1124163-8210684.9±0.6[16]ASGAGTGACGGCTTTGTAGTCC168-1483Vibrio
parahaemoliticustdhTdh199FGGTACTAAATGGCTGACATCX54341601-58225181.6±0.3[17]RCCACTACCACTCTCATATGC351-370EmeticBacillus cereusces*ces-TMFGATGTTTGCGACGATGCAADQ3608258689-87076580.4±0.1This studyRCTTTCGGCGTGATACCCATT8793-87344Salmonella spp.invA*StyinvaJHO-2-rightTCGTCATTCCATTACCTACCM90846167-18611981.3±0.4[5]JHO-3-leftAAACGTTGAAAAACTGAGGA285-234Clostridium
perfringenscpeGAP11GGTTCATTAATTGAAACTGGTGX81849583-60415478.3±0.4[18]12AACGCCAATCATATAAATTACAGC712-736Second and third runs5 ETEC (ST)STSTaFGCTAATGTTGGCAATTTTTATTTCTGTAM25607294-32119078.5±0.2[19]RAGGATTACAACAAAGTTCACAGCAGTAA483-456Plesiomonas
shigelloidesgyrBPSG237-FTTCCAGTACGAGATCCTGGCTAAAJ300545237-2596883.1±0.2[13]304-RTGAATCGACACGCCAGAGTTC304-2846 EAggECaggR*aggR-ZFCAGAATCGTCAGCATCAGCTACAZ18751432-4549779.5±0.3This studyRGATGCCCTGATGATAATATACGGAA358-382EIEC &
Shigella spp.virAvirAFCTGCATTCTGGCAATCTCTTCACAD264681589-162221582.4±0.3[20]RTGATGAGCTAACTTCGTAAGCCCTCC1813-17887Aeromonas
hydrophilaahh1*AHH1FGCCGAGCGCCCAGAAGGTGAGTTCP0004621653360-8213389.8±0.4[21]RGAGCGGCTGGATGCGGTTGT1653492-73ETEC (LT)LTLT1AGCAGGTTTCCCACCGGATCACCAS60731613-63613282.0±0.3[22]2GTGCTCAGATTCTGGGTCTC744-7258Providencia
alcalifaciensgyrBPAG38FTCTGCACGGTGTGGGTGTTAJ30054738-567381.0±0.2[13]110RACCGTCACGGCGGATTACT110-92EnterotoxigenicB. cereusnheB*SGF3GCACTTATGGCAGTATTTGCAGCDQ1532572101-212315282.7±0.4[23]R3GCATCTTTTAAGCCTTCTGGTC2252-22319Yersinia
enterocoliticayadA*yadA-XFCCAGAACCAATTGCAATGCCTX138821564-154310081.6±0.2This studyY.
pseudotuberculosisRCTTTAAACAGCTTGTTCCAGCCA1465-148781.1±0.3Campylobacter
coliceuE*CCceuE825FACGCGCACAAGGCATACTTX888493513-35319177.6±0.3This study915RCCAGTATTCAGGATCAAGATAAATGATTT3603-357510Vibrio
choleraehlyAhlyA2272-FAGCAGCGTGTGGGACAAGAX517462272-22917182.4±0.1[13]2272-FGCGGACCCTAATGCATCAAT2342-2323Vibrio
parahaemoliticustrhTrh250-FGGCTCAAAATGGTTAAGCGDQ359748256-27425081.1±0.1[17]251-RCATTTCCGCTCTCATATGC505-487Singl PCREHEC (Stx 1)Stx1JMS1FGTCACAGTAACAAACCGTAACAEF441598509-4889581.1±1.0[12]RTCGTTGACTACTTCTTATCTGGA415-437Singl PCREHEC (Stx 2)Stx2JMS2FCGACCCCTCTTGAACATAEF441616140-15710881.7±0.3[12]RGATAGACATCAAGCCCTCGT247-228a: Average ± standard
deviation ofTm values of 10 tests: b: *:new selected or designed
primer


### 2.3. Duplex SG-PCR with Feces

Feces
(1 g) from 5 patients were weighed aseptically from the mass sample collected
for virological inspection, placed into sterile tubes, and homogenized with 9 mL
of distilled water.  Then, 200μL of
stool suspension was treated with a QIAamp DNA Stool Mini kit.  For real-time PCR, we used SYBR Premix EX Taq (Takara, Japan), 32 glass
capillary tubes, and a LightCycler instrument (Roche Diagnostics, Mannheim,
Germany) as described by the manufacturer. Duplex SG-PCR was performed using 32
glass capillary tubes with 4 groups of 2 primer sets on the LC instrument for
each run. Analysis of each group of primer pairs was made in 8 glass capillary
tubes; each of which included 1 negative DNA control consisting of PCR-grade
water, 2 positive controls, and template DNA from 5 feces. The first run of
duplex SG-PCR was analyzed using 4 primer sets selected from 11 primer sets
described in our previous reports [10, 13]. The newly first run primer set including eae plus FemB, AB plus EAST1,
Tdh plus Ces-TM, and Styinva plus GAP (see Table 2) was used for analysis of cases
19 to 21. The second run was analyzed using 4 primer sets selected from the
following primer sets: LT plus AHH1, STa plus PSG, aggR-Z plus virA, SG plus
PAG and the third run using yadA-X plus CCceuE, and hlyA plus Trh. The eaeA-positive
samples were analyzed by simple PCR using primers JMS1 and JMS2. Each reaction
tube contained 10 μL of SYBR Premix EX
Taq, 6.8 μL of PCR-grade H2O, 0.4 μL of both forward and reverse
primers (10 μM) for the target gene of two foodborne pathogens, and 2 μL of
template DNA in a 20 μL PCR mixture. The assay cycling profile was 95C° for 10 minutes,
followed by 30 cycles of denaturation at 95C° for 5 seconds and then annealing
at 60C° for 20 seconds. Fluorescence signals were measured once per cycle at the
end of the extension step. After PCR amplification, a melting temperature
curve analysis was done. Next, the LightCycler PCR products were cooled to
65C° and then heated to 95C° at a rate of 0.1C° per second. The fluorescence
signals obtained were continuously monitored to confirm amplification
specificity during 1 hour of analysis. The products’ melting temperature peaks
were calculated by performing 10 or more assays per sample and were based on
the initial fluorescence curve found by plotting the negative derivative of
fluorescence over temperature versus temperature. To quantify target bacteria
in feces, DNA samples extracted with the QIAamp DNA Stool Mini kit from target
bacteria were used to form a standard curve. Two microliters of a serial
10-fold dilution of DNA (Easy Dilution from Takara, Japan) were prepared and
analyzed under the conditions specified above.


### 2.4. Duplex SG-PCR Analysis in 21 Foodborne Outbreaks

21 foodborne
outbreak cases examined by duplex SG-PCR in Shimane Prefecture, Japan from 2002
to 2007 are shown in Table3.Table 3
Epidemiological investigations in 21
food—borne outbreaks examined by SG-PCR and bacteriological cultures in
Shimane Prefecture, Japan.
Case No.Date ocurred (day/mo/yr)Days for examination after occurrenceInfected groupSource of infection (suspected source)No. of patients/totalNo. of examined patientsCausative pathogensStool samples (No. of positive/ examined samples)SG-PCRIsolation1st test2nd test3rd testFinal testTotal14-Oct-026School excursion in a
mountain areaStream watera23/3322*EPEC O:125, O:166, O:UT *astA-positive E. coli O:1, O:UT1/7——4/227/225/22203-Sep-033Protective care schoolCatering box lunch22/4610astA-positive E. coli O:18,1/5——6/106/103/10O:20, O:114, O:159,
O:UT[Norovirus6/7]301-Oct-032Celebration in a companyCatering box lunch437/135412*C. perfringens O:13, O:165/5——7/127/1210/12411-Jun-046Camping group of high schoolGrilled meat (beef, bovine
intestinal meat)4/84C. jejuni1/4——1/81/85/8512,13-Jun-046 ~ 79 citizen groups in
ChophouseGrilled meat (beef, bovine
intestinal meat)30/UN12C. jejuni4/5——8/128/1210/12617-Jun-045Cooking practise in a high
schoolShelf-cooked lunch (salada
mixed chicken)31/4120*C. jejuni4/5——12/1412/1417/20707-Jul-041Citizen in Chinese
restaurantFried riceb6/66*B. cereus1/1——2/62/62/6811-Oct-043Sport club in a high schoolShelf-cooked lunch26/476*C. perfringens O:16, OUT1/5——3/63/64/695~7-Nov-045 ~ 7RestaurantUnknown55C. jejuni2/5——2/52/510UnknownSeveral days
(19-Jur-05)NurseryUnknown24/7322*EHEC O268/228/228/22[Norovirus20/22]1128~30-Sep-051 ~
3Prisoners in a
prisonShelf-cooked
mealc113/60061astA -positive E. coli——14/1414/1441/46C. perfringens (sporadic case)1/51/46122~6-Oct-051 ~ 5Elementary and
high school childrenUnknown (School
lunch)39/9439astA -positve E. coli5/5——5/5IMfEPEC2/5IMA. hidrophila1/51/16C. jejuni (sporadic case)1/5IM1328~30-May-060 ~ 2Citizens at
restaurantLunch (pilaf and
scrambled aggd)27/3427*S. aureus2/5——2/54/8astA-positve E. coli1/5144-Jul-060Boarder of high
schoolCatering box lunch34/5134*C.
perfringens5/5——8/88/819/501516-Aug-061Citizens at
restaurantFried rice15/3415*B. cereus1/4——1/42/41623~29-Aug-062 ~ 8Boarder of
training high schoolSupper
(contaminated sliced cabbagee)19/4318*C. jejuni3/5——6/98/99/14astA -positve E. coli4/55/9IM172-Sep-063Citizens in
Buddhist serviceCatering box lunch14/494V.
parahaemolyticus4/5——4/64/63/61822-Dec-065Citizens at
restaurantSupper (chiken)12/128*C. jejuni3/5——4/94/94/10194-Jul-076Citizens at
restaurantSupper (chiken)7/117*C. jejuni1/2——1/22/32021-Oct-071Citizens at
restaurantSupper7/137*EPEC2/5—4/5IMP. shigelloides2/52/52129-Nov-071Citizens at
restaurantSupper (raw
chiken liver)8/87*C. jejuni3/5——4/75/74/7astA -positve E. coli1/51/7Total54/93111/191160/27658.1%58.1%58.0%a: EPEC O : 166, O : UT andastA -positive E. coli O : 27, O : UT strains were isolated from stream water drunk by
patients in case 1., b: B cereus was isolated from cooked pork in case
7. c: astA genes were detected from 5 food samples in case 11., d: S. aureus was isolated from pilaf
and scrambled egg in case 13., e: C. jejuni specific gene was detected
from 5 food samples in case 17. f: Impossible isolation. *: 14 cases examined
by SG-qPCR and viable cell count.


## 2.1. Bacterial Strains

The 27 foodborne bacteria used in this
study areE. coli (enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)), Shigellasonnei, Salmonella Enteritidis, Yersinia
enterocolitica, Yersinia pseudotuberculosis, Providencia alcalifaciens, Plesiomonas
shigelloides, Campylobacter jejuni, C. coli, Vibrio cholerae, TDH-positive V.
parahaemolyticus, TRH-positive V. parahaemolyticus, Aeromonas hydrophila, Staphylococcus aureus, emetic Bacillus cereus, enterotoxigenic B. cereus, and Clostridium perfringens (Table 1). Bacterial cultures and viable-cell counting were
described in a previous report [10]. For template DNA of each foodborne
pathogen as a PCR control, 200 μL of each bacterial culture (108 CFU/mL) was treated with a QIAamp DNA Stool Mini kit (Qiagen) in the same
procedure as the following stool treatments.Table 1
Bacterial strains assayed by SYBR Green I PCR
Bacterial strainsSourcesePCR results with each primer set (see Table2)eaeJMS1JMS2LTSTaEAST-1aggR-ZvirAStyinvayadA-XPAGPSGABCC ceuEhlyAtdhtrhAHH1FemBces-TMSGGAPEscherichia coli -EPECO55(eaeA)EC-2736b+−−−−−−−−−−−−−−−−−−−−−E. coli -EPEC
O153 (eaeA and astA)EC-2649b+−−−−+−−−−−−−−−−−−−−−−E. coli -EHEC O26:H11(Stx1)SE-02005++−−−−−−−−−−−−−−−−−−−−E. coli -EHEC O157:H7 (Stx2)SE020025+−+−−−−−−−−−−−−−−−−−−−E. coli -EHEC O157:H7
(Stx1 and Stx2)SE-02027+++−−−−−−−−−−−−−−−−−−−E. coli -ETEC O148
(LT, ST and astA)EC-3515b−−−+++−−−−−−−−−−−−−−−−E. coli -ETEC O169
(ST and astA)EC-4725b−−−−++−−−−−−−−−−−−−−−−E. coli- EAEC O111 (aggR and astA)EC-4131b−−−−−++−−−−−−−−−−−−−−−E. coli -EIEC O124:HNM (virA)EA32a−−−−−−−+−−−−−−−−−−−−−−Shigella sonneiI00031−−−−−−−+−−−−−−−−−−−−−−Salmonella EnteritidisSal-2339−−−−−−−−+−−−−−−−−−−−−−Yersinia enterocolitica O3/B4Pa241−−−−−−−−−+−−−−−−−−−−−−Y. pseudotuberculosis O4bSP988−−−−−−−−−+−−−−−−−−−−−−Providencia alcalifaciensNIID124C−−−−−−−−−−+−−−−−−−−−−−Plesiomonas shigelloidesNIID123C−−−−−−−−−−−+−−−−−−−−−−Campylobacter jejuniSC 009−−−−−−−−−−−−+−−−−−−−−−Campylobacter coliSC 011−−−−−−−−−−−−−+−−−−−−−−Vibrio cholerae O1ATCC14035−−−−−−−−−−−−−−+−−−−−−−V. cholerae O139NIID63-93C−−−−−−−−−−−−−−+−−−−−−−V. cholerae non- O1SVP84−−−−−−−−−−−−−−+−−−−−−−V. parahaemoliticus O3:K6 (tdh)SVP02−−−−−−−−−−−−−−−+−−−−−−V. parahaemoliticus O3:K6 (trh)NIIDK4C−−−−−−−−−−−−−−−−+−−−−−Aeromonas hydrophila O1ATCC7966−−−−−−−−−−−−−−−−−+−−−−Staphylococcus aureusSS 05e−−−−−−−−−−−−−−−−−−+−−−EmeticBacillus cereusNo.127e−−−−−−−−−−−−−−−−−−−++−EnterotoxigenicB. cereusNo.1e−−−−−−−−−−−−−−−−−−−−+−Clostridium perfringensH2d−−−−−−−−−−−−−−−−−−−−−+Strain kindly donated by K. Sugiyamaa,
Shizuoka Prefectural Institute of Public Health, Shizuoka; J. Yatsuyanagib, Akita
Prefectural Institute of Public Health, Akita; M. Tamura and E. Arakawac,
Ofther strains except for ATCC numbers are our own
collections.


## 2.2. Primer Design

The
22 primer pairs used in this study for the detection ofE. coli (EIEC, EPEC, EHEC, ETEC, and EAEC), Salmonellaenterica, Shigella spp., Y. enterocolitica, Y. pseudotuberculosis, P. alcalifaciens, C. jejuni,
C. coli, V. cholerae,  V. parahaemolyticus, A. hydrophila, P. shigelloides, S. aureus, C. perfringens,
and B. cereus
were
described in our previous reports [10, 13] for cases 1 to 19. The newly designed 22
primer pairs listed in Table 2 were used for cases 19 to 21. In this study, 10
primer pairs (marked with * in Table 2) were newly designed or selected from
earlier publications (see Table 2 references). The 4 primer pairs (ces, yadA-X,
CCceuE, and aggR-Z) were newly designed. The ces primer was constructed from cereulide synthetase gene of emetic B. cereus [4], the
yadA-X primer from yadA gene on the
plasmid present in virulent Yersinia spp. [24], the CCceuE primer from ceuE gene
encoding of a lipoprotein component of a binding-protein-dependent transport
system for the siderophore enterochelin of C.
coli [25], and the aggR-Z primer from aggR gene encoding of a transcriptional activator
for EAEC aggregative adherence fimbria I expression [26]. To
determine the specific primers ces,
yadA-X, CCceuE, and aggR-Z, the genes of ces, yadA, ceuE, and aggR that were expected to be unique were selected with the Basic
Local Alignment Search Tool (BLAST) program within GenBank and were designed by Biosearch Technologies Inc. (USA). Other primer pairs were
those used in earlier publications (see Table 2 references). All
oligonucleotide primers were synthesized by Invitrogen (Yokohama, Japan)
or Biosearch Technologies Inc. (USA).Table 2
22 pairs of specific primers for SYBR Green I PCR
Primer set for duplex PCRSpecies and subgroupsTarget genePCR primersGenBank accession no.locationProduct size (bp)Tm valuesaReferencesNameForward or reversprimers' sequences (5' - 3')First run1Escherichia
colieaeA*beaeF2CATTGATCAGGATTTTTCTGGTGATAZ11541899-92410683.2±0.2[14]EPEC and EHECRCTCATGCGGAAATAGCCGTTA1000-979Staphylococcus
aureusfemB*FemBfwAATTAACGAAATGGGCAGAAACAAF106850277-2999380.8±0.3[15]rvTGCGCAACACCCTGAACTT370-3512Campylobacter
jejuniC. jejuni- specific DNA*ABFCTGAATTTGATACCTTAAGTGCAGCAL1111683811358679.1±0.4[8]DNARAGGCACGCCTAAACCTATAGCT381185EAECastAEAST-1SGCCATCAACACAGTATATCCL1124163-8210684.9±0.6[16]ASGAGTGACGGCTTTGTAGTCC168-1483Vibrio
parahaemoliticustdhTdh199FGGTACTAAATGGCTGACATCX54341601-58225181.6±0.3[17]RCCACTACCACTCTCATATGC351-370EmeticBacillus cereusces*ces-TMFGATGTTTGCGACGATGCAADQ3608258689-87076580.4±0.1This studyRCTTTCGGCGTGATACCCATT8793-87344Salmonella spp.invA*StyinvaJHO-2-rightTCGTCATTCCATTACCTACCM90846167-18611981.3±0.4[5]JHO-3-leftAAACGTTGAAAAACTGAGGA285-234Clostridium
perfringenscpeGAP11GGTTCATTAATTGAAACTGGTGX81849583-60415478.3±0.4[18]12AACGCCAATCATATAAATTACAGC712-736Second and third runs5 ETEC (ST)STSTaFGCTAATGTTGGCAATTTTTATTTCTGTAM25607294-32119078.5±0.2[19]RAGGATTACAACAAAGTTCACAGCAGTAA483-456Plesiomonas
shigelloidesgyrBPSG237-FTTCCAGTACGAGATCCTGGCTAAAJ300545237-2596883.1±0.2[13]304-RTGAATCGACACGCCAGAGTTC304-2846 EAggECaggR*aggR-ZFCAGAATCGTCAGCATCAGCTACAZ18751432-4549779.5±0.3This studyRGATGCCCTGATGATAATATACGGAA358-382EIEC &
Shigella spp.virAvirAFCTGCATTCTGGCAATCTCTTCACAD264681589-162221582.4±0.3[20]RTGATGAGCTAACTTCGTAAGCCCTCC1813-17887Aeromonas
hydrophilaahh1*AHH1FGCCGAGCGCCCAGAAGGTGAGTTCP0004621653360-8213389.8±0.4[21]RGAGCGGCTGGATGCGGTTGT1653492-73ETEC (LT)LTLT1AGCAGGTTTCCCACCGGATCACCAS60731613-63613282.0±0.3[22]2GTGCTCAGATTCTGGGTCTC744-7258Providencia
alcalifaciensgyrBPAG38FTCTGCACGGTGTGGGTGTTAJ30054738-567381.0±0.2[13]110RACCGTCACGGCGGATTACT110-92EnterotoxigenicB. cereusnheB*SGF3GCACTTATGGCAGTATTTGCAGCDQ1532572101-212315282.7±0.4[23]R3GCATCTTTTAAGCCTTCTGGTC2252-22319Yersinia
enterocoliticayadA*yadA-XFCCAGAACCAATTGCAATGCCTX138821564-154310081.6±0.2This studyY.
pseudotuberculosisRCTTTAAACAGCTTGTTCCAGCCA1465-148781.1±0.3Campylobacter
coliceuE*CCceuE825FACGCGCACAAGGCATACTTX888493513-35319177.6±0.3This study915RCCAGTATTCAGGATCAAGATAAATGATTT3603-357510Vibrio
choleraehlyAhlyA2272-FAGCAGCGTGTGGGACAAGAX517462272-22917182.4±0.1[13]2272-FGCGGACCCTAATGCATCAAT2342-2323Vibrio
parahaemoliticustrhTrh250-FGGCTCAAAATGGTTAAGCGDQ359748256-27425081.1±0.1[17]251-RCATTTCCGCTCTCATATGC505-487Singl PCREHEC (Stx 1)Stx1JMS1FGTCACAGTAACAAACCGTAACAEF441598509-4889581.1±1.0[12]RTCGTTGACTACTTCTTATCTGGA415-437Singl PCREHEC (Stx 2)Stx2JMS2FCGACCCCTCTTGAACATAEF441616140-15710881.7±0.3[12]RGATAGACATCAAGCCCTCGT247-228a: Average ± standard
deviation ofTm values of 10 tests: b: *:new selected or designed
primer


## 2.3. Duplex SG-PCR with Feces

Feces
(1 g) from 5 patients were weighed aseptically from the mass sample collected
for virological inspection, placed into sterile tubes, and homogenized with 9 mL
of distilled water.  Then, 200μL of
stool suspension was treated with a QIAamp DNA Stool Mini kit.  For real-time PCR, we used SYBR Premix EX Taq (Takara, Japan), 32 glass
capillary tubes, and a LightCycler instrument (Roche Diagnostics, Mannheim,
Germany) as described by the manufacturer. Duplex SG-PCR was performed using 32
glass capillary tubes with 4 groups of 2 primer sets on the LC instrument for
each run. Analysis of each group of primer pairs was made in 8 glass capillary
tubes; each of which included 1 negative DNA control consisting of PCR-grade
water, 2 positive controls, and template DNA from 5 feces. The first run of
duplex SG-PCR was analyzed using 4 primer sets selected from 11 primer sets
described in our previous reports [10, 13]. The newly first run primer set including eae plus FemB, AB plus EAST1,
Tdh plus Ces-TM, and Styinva plus GAP (see Table 2) was used for analysis of cases
19 to 21. The second run was analyzed using 4 primer sets selected from the
following primer sets: LT plus AHH1, STa plus PSG, aggR-Z plus virA, SG plus
PAG and the third run using yadA-X plus CCceuE, and hlyA plus Trh. The eaeA-positive
samples were analyzed by simple PCR using primers JMS1 and JMS2. Each reaction
tube contained 10 μL of SYBR Premix EX
Taq, 6.8 μL of PCR-grade H2O, 0.4 μL of both forward and reverse
primers (10 μM) for the target gene of two foodborne pathogens, and 2 μL of
template DNA in a 20 μL PCR mixture. The assay cycling profile was 95C° for 10 minutes,
followed by 30 cycles of denaturation at 95C° for 5 seconds and then annealing
at 60C° for 20 seconds. Fluorescence signals were measured once per cycle at the
end of the extension step. After PCR amplification, a melting temperature
curve analysis was done. Next, the LightCycler PCR products were cooled to
65C° and then heated to 95C° at a rate of 0.1C° per second. The fluorescence
signals obtained were continuously monitored to confirm amplification
specificity during 1 hour of analysis. The products’ melting temperature peaks
were calculated by performing 10 or more assays per sample and were based on
the initial fluorescence curve found by plotting the negative derivative of
fluorescence over temperature versus temperature. To quantify target bacteria
in feces, DNA samples extracted with the QIAamp DNA Stool Mini kit from target
bacteria were used to form a standard curve. Two microliters of a serial
10-fold dilution of DNA (Easy Dilution from Takara, Japan) were prepared and
analyzed under the conditions specified above.


## 2.4. Duplex SG-PCR Analysis in 21 Foodborne Outbreaks

21 foodborne
outbreak cases examined by duplex SG-PCR in Shimane Prefecture, Japan from 2002
to 2007 are shown in Table3.Table 3
Epidemiological investigations in 21
food—borne outbreaks examined by SG-PCR and bacteriological cultures in
Shimane Prefecture, Japan.
Case No.Date ocurred (day/mo/yr)Days for examination after occurrenceInfected groupSource of infection (suspected source)No. of patients/totalNo. of examined patientsCausative pathogensStool samples (No. of positive/ examined samples)SG-PCRIsolation1st test2nd test3rd testFinal testTotal14-Oct-026School excursion in a
mountain areaStream watera23/3322*EPEC O:125, O:166, O:UT *astA-positive E. coli O:1, O:UT1/7——4/227/225/22203-Sep-033Protective care schoolCatering box lunch22/4610astA-positive E. coli O:18,1/5——6/106/103/10O:20, O:114, O:159,
O:UT[Norovirus6/7]301-Oct-032Celebration in a companyCatering box lunch437/135412*C. perfringens O:13, O:165/5——7/127/1210/12411-Jun-046Camping group of high schoolGrilled meat (beef, bovine
intestinal meat)4/84C. jejuni1/4——1/81/85/8512,13-Jun-046 ~ 79 citizen groups in
ChophouseGrilled meat (beef, bovine
intestinal meat)30/UN12C. jejuni4/5——8/128/1210/12617-Jun-045Cooking practise in a high
schoolShelf-cooked lunch (salada
mixed chicken)31/4120*C. jejuni4/5——12/1412/1417/20707-Jul-041Citizen in Chinese
restaurantFried riceb6/66*B. cereus1/1——2/62/62/6811-Oct-043Sport club in a high schoolShelf-cooked lunch26/476*C. perfringens O:16, OUT1/5——3/63/64/695~7-Nov-045 ~ 7RestaurantUnknown55C. jejuni2/5——2/52/510UnknownSeveral days
(19-Jur-05)NurseryUnknown24/7322*EHEC O268/228/228/22[Norovirus20/22]1128~30-Sep-051 ~
3Prisoners in a
prisonShelf-cooked
mealc113/60061astA -positive E. coli——14/1414/1441/46C. perfringens (sporadic case)1/51/46122~6-Oct-051 ~ 5Elementary and
high school childrenUnknown (School
lunch)39/9439astA -positve E. coli5/5——5/5IMfEPEC2/5IMA. hidrophila1/51/16C. jejuni (sporadic case)1/5IM1328~30-May-060 ~ 2Citizens at
restaurantLunch (pilaf and
scrambled aggd)27/3427*S. aureus2/5——2/54/8astA-positve E. coli1/5144-Jul-060Boarder of high
schoolCatering box lunch34/5134*C.
perfringens5/5——8/88/819/501516-Aug-061Citizens at
restaurantFried rice15/3415*B. cereus1/4——1/42/41623~29-Aug-062 ~ 8Boarder of
training high schoolSupper
(contaminated sliced cabbagee)19/4318*C. jejuni3/5——6/98/99/14astA -positve E. coli4/55/9IM172-Sep-063Citizens in
Buddhist serviceCatering box lunch14/494V.
parahaemolyticus4/5——4/64/63/61822-Dec-065Citizens at
restaurantSupper (chiken)12/128*C. jejuni3/5——4/94/94/10194-Jul-076Citizens at
restaurantSupper (chiken)7/117*C. jejuni1/2——1/22/32021-Oct-071Citizens at
restaurantSupper7/137*EPEC2/5—4/5IMP. shigelloides2/52/52129-Nov-071Citizens at
restaurantSupper (raw
chiken liver)8/87*C. jejuni3/5——4/75/74/7astA -positve E. coli1/51/7Total54/93111/191160/27658.1%58.1%58.0%a: EPEC O : 166, O : UT andastA -positive E. coli O : 27, O : UT strains were isolated from stream water drunk by
patients in case 1., b: B cereus was isolated from cooked pork in case
7. c: astA genes were detected from 5 food samples in case 11., d: S. aureus was isolated from pilaf
and scrambled egg in case 13., e: C. jejuni specific gene was detected
from 5 food samples in case 17. f: Impossible isolation. *: 14 cases examined
by SG-qPCR and viable cell count.


## 3. Results and Discussion

### 3.1. Duplex SG-PCR Procedures

We previously reported duplex SG-PCR assays for detection of 19 species of
foodborne pathogens using 22 primer pairs [10, 13]. After that, more accurate
duplex SG-PCR assays were designed by 10 more sensitive and specific primers including
6 primers (FemB, AB, ces-TM, Styinva, SG, and AHH1) selected from earlier
publications (see references in Table 2) and 5 new primers (eae, aggR-Z,
yadA-X, and CCceuE) constructed in this study. The new primer set was used for
cases 19 to 21. Real-time SG-PCR procedures using 22 primer pairs for the
detection of 15 bacterial species, including 5 E coli subgroups, were developed for the duplex assay. The primer
sequence, target, SG-PCR product size, Tm values (mean plus standard deviation from a range of 10 assays),
specificities, and references are summarized and listed in Tables 1 and 2. The
primer virA detects virA gene of Shigella spp. and EIEC; the primer eae
detects eaeA gene of EPEC and EHEC,
and the primer EAST-1 detects astA gene of EAEC and ETEC. Primer hlyA detected hlyA gene of V. cholerae strains O1 and
O139 as well as non-O1 strains. The primer SG for the detection of nheB (nonhemolytic enterotoxin B) gene
of B. cereus cross-reacts with
enterotoxigenic and emetic strains and the primer ces-TM detects cereulide synthetase
gene of emetic strain of B. cereus. The nheB and ces gene positive strains were identified with emetic strains and
the nheB gene positive and ces gene negative strains with enterotoxigenic
strains. A new primer yadA-X for Yersinia adhesion reacts with virulent Y. enterocolitica and Y. pseudotuberculosis, but not with
nonpathogenic strains of Yersinia spp.
(data not shown).  Other primers,
including new primers aggR-Z and CCceuE, specifically detect each gene of EAEC
and C. coli. Food-borne Outbreak
Investigation Report (http://www.mhlw.go.jp/topics/syokuchu/), Ministry of
Health, Labor and Welfare, Japan during 2005 to 2007 shows that 97% of foodborne
outbreaks were caused by the following 7 species of foodborne pathogens: S. enterica (58.3%), C. jejuni (15.2%), TDH-producing V. parahaemolyticus (8.3%),
S. aureus (7.2%), C. perfringens (3.6%), emetic B. cereus (1.6%), EHEC
(2.9%), and other virulent E. coli (2.1%) which include astA-positive E. coli which is a strain of E. coli that does not possess any
diarrheagenic characteristics except the EAEC heat-stable toxin 1 (EAST1) gene
and is frequently isolated in diarrhea outbreaks [27]. Using of 4 primer sets
of 2 primer pairs, including newly selected or designed 6 primer pairs, for the
detection of 7 main foodborne pathogens and astA-positive E. coli in the first run of duplex
SG-PCR brought out the comprehensive, rapid, and
sensitive detection of
causative pathogens in foodborne pathogens to cases 19 to 21 (Table 2 and
Figures 1 and 2). The second run of duplex SG-PCR used 4 primer sets and the
final run utilized 2 primer sets selected from the remaining 4 primer pairs. The
primers JMS1 and JMS2 were used for the single PCR detection of stx1 and/or stx2 genes from the eaeA gene-positive samples for the confirmation of EHEC. Figures 1 and 2 show the Tm curves of the duplex
SG-PCR products of the template DNA samples in each run. In duplex SG-PCR assay
with two primer pairs, each PCR product was generated with a different Tm curve. These could be
resolved in a LightCycler by using Tm curve analysis when a target bacterium was present in the reaction
tube.Melting
curve analysis of duplex SYBR Green I PCR products in the first run using four
primer sets: FemB plus eaeA, AB plus EAST1, ces plus tdh, and GAP plus Styinva.
(a)(b)(c)(d)Melting
curve analysis of duplex SYBR Green I PCR products in the second run using four
primer sets: ST plus PSG, aggR plus virA, LT plus AHH1, and PAG plus SG; the
third run using two primer sets: CCcesE plus yadA and trh plus hlyA; simple PCR
with primers JMS 1 and JMS2.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 3
The relationship between CFU and DNA copy of
foodborne pathogens in 71 foodborne pathogens-positive feces in 14 foodborne
outbreak cases examined by viable cell counting.


### 3.2. Using Duplex SG-PCR for Identification of the Causative Agent in 21 Foodborne Outbreaks

Table3 shows epidemiological and clinical
investigations in 21 foodborne outbreaks examined by duplex SG-PCR analysis in
Shimane Prefecture, Japan from 2002 to 2007.
From samples of feces, we used a combination of duplex SG-PCR assay with
DNA extraction using a QIAamp DNA Stool Mini kit.  The SG-PCR assay is rapid, specific, and
sensitive as a detection technique. The DNA extraction of 5 stool specimens
with the QIAamp DNA Stool Mini kit was carried out within 1 hour and it
effectively removed inhibitors present in feces. The duplex SG-PCR assay was
also carried out within 1 hour. The 7 species (listed previously) of foodborne
bacteria, which included 3 groups of E.
coli, were detected from 111 (58.1%) of 191 feces in 21 cases by duplex
SG-PCR. Then these causative agents were isolated and identified after 2 to 4
days. With the exception of two cases (cases 10 and 11), the first run of
duplex SG-PCR confirmed the presence of a pathogen in 54 (58.1%) of 93 feces in
19 (90.5%) cases within 2 hours. The exceptions were case 10 where a
confirmation test was necessary to detect the eaeA gene of EHEC O26 and case 11 where astA-positive E. coli was
detected on the third run. In the first run, DNA samples extracted from 5 feces
(1, 3, 4, or 7 feces in 6 cases) of symptomatic patients were used and the
causative pathogens were detected from 1 to 5 samples: 1 (in 8 cases: 1, 2, 4,
7, 8, 15, 19, and 21), 2 (in 3 cases: 9, 13, and 20), 3 (in 3 cases: 16, 18,
and 21), 4 (in 3 cases: 5, 6, and 17), and 5 samples (in 3 cases: 3, 12, and
14). Then the causative pathogens were later isolated in a routine laboratory. In
cases 11 and 12, C. perfringens or C. jejuni was detected by duplex SG-qPCR
with more than 105 CFU/g feces from only 1 sample and C. perfringens was then also isolated
from only 1 of 46 samples and C. jejuni from only 1 of 16 samples by culture method. Therefore, the infections with
both these pathogens were determined to be sporadic cases and they were
immediately eliminated as causative pathogens in cases 11 and 12. It was
confirmed that duplex SG-PCR analysis of 5 feces collected from symptomatic
patients was ultimately the most effective screening method for foodborne
pathogens in foodborne outbreaks [10, 13].Duplex SG-PCR
rapidly and accurately demonstrated that 12 (57.1%) of 21 cases were caused
with a single foodborne pathogen such asC. jejuni (6 cases), C. perfringens (3
cases), B. cereus (2 cases), and TDH-producing V. parahaemolyticus (one case). There were also 7 (33.3%) cases
with plural foodborne bacterial pathogens (such as astA-positive E. coli, EPEC, C. jejuni, C. perfringens, A. hydrophila, and P. shigelloides) and 2 (9.5%)
cases with foodborne bacterial pathogens (astA-positive E. coli or EHEC O:26) and norovirus. In
cases 2 and 10, although detection of norovirus is out of the scope of our work,
norovirus and foodborne bacterial pathogens were concomitantly detected by
conventional PCR analysis in our virological laboratory. In case 2 in which
norovirus was detected in 6 of 7 feces, the astA gene of EAEC was detected from 7 of
10 feces and then astA-positive E. coli strains were isolated from 6
samples. In case 10 in which norovirus was detected from 20 of 22 feces, the eae gene of EPEC or EHEC was detected
from 8 of 22 feces and EHEC O26 strains were isolated from 8 of 22 feces. In 7
cases (cases 1, 11, 12, 13, 16, 20, and 21), the pathogenic E. coli strains belonging to astA-positive E. coli and/or EPEC were concomitantly detected with other foodborne bacterial
pathogens. In case 1, the eae gene of
EPEC or EHEC was detected from 4 of 22 feces and the astA gene of EAEC was detected in 3 other feces.  However, duplex SG-PCR could not detect other
virulent genes, including the stx1
and stx2 genes of EHEC. Then EPEC
strains were later isolated from 5 feces and astA-positive E. coli from 4 other feces. In case 12, the astA gene of EAEC was detected in all 5 feces and the eae gene of EPEC or EHEC in 2 feces, but duplex SG-PCR could not detect
other E. coli virulent genes. The
subsequent bacteriological examination could not isolate pathogenic E. coli among nonpathogenic E. coli flora.  In case 16, the C. jejuni specific gene was detected in 6 of 9 feces and the astA gene of EAEC was detected in 5
feces (both genes from 3 feces). C.
jejuni strains were then isolated from 9 of 14 feces, but we were not able
to isolate the pathogenic E. coli strain among nonpathogenic E. coli flora.  In cases 19 to 21 analyzed
improved real-time PCR using 8 primers for the detection of 7 main foodborne
bacteria and astA-positive E. coli, C. jejuni, EPEC, or astA-positive E. coli
were detected
from 1 to 3 fecal samples on the first run and the absence of the other main
foodborne bacteria in the analyzed samples was readily confirmed. In case 20,
the eae gene of EPEC or EHEC was
detected from 2 of 5 fecal samples on the first run and the gyrB gene of P. shigelloides
was
detected separately from other 2 fecal samples on the second run. Then P. shigelloides strains were isolated
from 2 feces, but isolation of the EPEC strain was very difficult due to the
presence of large nonpathogenic E. coli flora in the feces.In almost all
cases, the duplex SG-PCR assay first run detected these causative agents from more
than one of the five feces. Then, in almost all cases, the presence of a
causative agent (presumed from duplex SG-PCR assay) was confirmed by the
results of the final SG-PCR assay run and the bacteriological cultivation of
additional feces. These findings confirmed that for foodborne outbreaks duplex
SG-PCR is a useful tool for the rapid detection of both single and multiple
pathogens.


### 3.3. Quantification of the Causative Agent in 14 Foodborne
Outbreak Cases

Figure2 shows the relationship
between CFU and DNA copy of foodborne pathogens using SG-quantitative PCR
(qPCR) assay in 71 feces from 14 cases examined by viable cell counting.  There was no correlation (r2=0.1183) between CFU and DNA copy of foodborne pathogens in feces, although
almost all pathogens were detected by SG-PCR from feces registering more than
103 CFU/g by viable cell counting. By using SG- qPCR assay
combined with DNA extraction using the QIAamp DNA Stool Mini kit, Bibbal et
al. [28] reported a significant correlation between CFU and DNA copy of ampicillin-resistant Enterobacteriaceae in swine feces. Fu et
al. [29] reported a significant correlation
between CFU and DNA copy of Lactobacillus and total anaerobic bacteria in dog feces but found no correlation between CFU
and DNA copy of C. perfringens. Although accurate quantifications of foodborne
pathogens, including C. jejuni and C. perfringens, in feces were not
completely performed by SG-qPCR in this study, the presence of any foodborne
pathogens at more than 103 CFU/g feces was certainly confirmed by
melting curve analysis. There are two major problems for these
differences. One cause is different sample preparation that was used for CFU
from the feces stored in the transport medium and for qPCR using the mass sample collected for virological
inspection. Another cause is the approach used to construct the standard
curves that were prepared from pure bacterial cultures. These curves do not
relate with the “real” situation of a bacterial quantification in a
faecal sample and can in part explain the absence of correlation between CFU
and DNA copy of foodborne pathogens in faeces.In our routine bacteriological diagnostic laboratory, we used duplex SYBR
Green I PCR assay combined with DNA extraction via QIAamp DNA Stool Mini kit
for the detection of foodborne bacteria from 21 foodborne outbreak cases. The
causative bacteria were detected in almost all cases in 2 hours or less. The
first run was for the detection of 8 main foodborne bacteria and the second run
was for the detection of other unusual suspect bacteria. The results proved
that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, duplex
SG-PCR assay is not only very useful, but is also economically viable for one-step
differentiation of causative bacteria in fecal specimens obtained from
symptomatic patients. This then allows for effective diagnosis and
management of foodborne outbreak.


## 3.1. Duplex SG-PCR Procedures

We previously reported duplex SG-PCR assays for detection of 19 species of
foodborne pathogens using 22 primer pairs [10, 13]. After that, more accurate
duplex SG-PCR assays were designed by 10 more sensitive and specific primers including
6 primers (FemB, AB, ces-TM, Styinva, SG, and AHH1) selected from earlier
publications (see references in Table 2) and 5 new primers (eae, aggR-Z,
yadA-X, and CCceuE) constructed in this study. The new primer set was used for
cases 19 to 21. Real-time SG-PCR procedures using 22 primer pairs for the
detection of 15 bacterial species, including 5 E coli subgroups, were developed for the duplex assay. The primer
sequence, target, SG-PCR product size, Tm values (mean plus standard deviation from a range of 10 assays),
specificities, and references are summarized and listed in Tables 1 and 2. The
primer virA detects virA gene of Shigella spp. and EIEC; the primer eae
detects eaeA gene of EPEC and EHEC,
and the primer EAST-1 detects astA gene of EAEC and ETEC. Primer hlyA detected hlyA gene of V. cholerae strains O1 and
O139 as well as non-O1 strains. The primer SG for the detection of nheB (nonhemolytic enterotoxin B) gene
of B. cereus cross-reacts with
enterotoxigenic and emetic strains and the primer ces-TM detects cereulide synthetase
gene of emetic strain of B. cereus. The nheB and ces gene positive strains were identified with emetic strains and
the nheB gene positive and ces gene negative strains with enterotoxigenic
strains. A new primer yadA-X for Yersinia adhesion reacts with virulent Y. enterocolitica and Y. pseudotuberculosis, but not with
nonpathogenic strains of Yersinia spp.
(data not shown).  Other primers,
including new primers aggR-Z and CCceuE, specifically detect each gene of EAEC
and C. coli. Food-borne Outbreak
Investigation Report (http://www.mhlw.go.jp/topics/syokuchu/), Ministry of
Health, Labor and Welfare, Japan during 2005 to 2007 shows that 97% of foodborne
outbreaks were caused by the following 7 species of foodborne pathogens: S. enterica (58.3%), C. jejuni (15.2%), TDH-producing V. parahaemolyticus (8.3%),
S. aureus (7.2%), C. perfringens (3.6%), emetic B. cereus (1.6%), EHEC
(2.9%), and other virulent E. coli (2.1%) which include astA-positive E. coli which is a strain of E. coli that does not possess any
diarrheagenic characteristics except the EAEC heat-stable toxin 1 (EAST1) gene
and is frequently isolated in diarrhea outbreaks [27]. Using of 4 primer sets
of 2 primer pairs, including newly selected or designed 6 primer pairs, for the
detection of 7 main foodborne pathogens and astA-positive E. coli in the first run of duplex
SG-PCR brought out the comprehensive, rapid, and
sensitive detection of
causative pathogens in foodborne pathogens to cases 19 to 21 (Table 2 and
Figures 1 and 2). The second run of duplex SG-PCR used 4 primer sets and the
final run utilized 2 primer sets selected from the remaining 4 primer pairs. The
primers JMS1 and JMS2 were used for the single PCR detection of stx1 and/or stx2 genes from the eaeA gene-positive samples for the confirmation of EHEC. Figures 1 and 2 show the Tm curves of the duplex
SG-PCR products of the template DNA samples in each run. In duplex SG-PCR assay
with two primer pairs, each PCR product was generated with a different Tm curve. These could be
resolved in a LightCycler by using Tm curve analysis when a target bacterium was present in the reaction
tube.Melting
curve analysis of duplex SYBR Green I PCR products in the first run using four
primer sets: FemB plus eaeA, AB plus EAST1, ces plus tdh, and GAP plus Styinva.
(a)(b)(c)(d)Melting
curve analysis of duplex SYBR Green I PCR products in the second run using four
primer sets: ST plus PSG, aggR plus virA, LT plus AHH1, and PAG plus SG; the
third run using two primer sets: CCcesE plus yadA and trh plus hlyA; simple PCR
with primers JMS 1 and JMS2.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 3
The relationship between CFU and DNA copy of
foodborne pathogens in 71 foodborne pathogens-positive feces in 14 foodborne
outbreak cases examined by viable cell counting.


## 3.2. Using Duplex SG-PCR for Identification of the Causative Agent in 21 Foodborne Outbreaks

Table3 shows epidemiological and clinical
investigations in 21 foodborne outbreaks examined by duplex SG-PCR analysis in
Shimane Prefecture, Japan from 2002 to 2007.
From samples of feces, we used a combination of duplex SG-PCR assay with
DNA extraction using a QIAamp DNA Stool Mini kit.  The SG-PCR assay is rapid, specific, and
sensitive as a detection technique. The DNA extraction of 5 stool specimens
with the QIAamp DNA Stool Mini kit was carried out within 1 hour and it
effectively removed inhibitors present in feces. The duplex SG-PCR assay was
also carried out within 1 hour. The 7 species (listed previously) of foodborne
bacteria, which included 3 groups of E.
coli, were detected from 111 (58.1%) of 191 feces in 21 cases by duplex
SG-PCR. Then these causative agents were isolated and identified after 2 to 4
days. With the exception of two cases (cases 10 and 11), the first run of
duplex SG-PCR confirmed the presence of a pathogen in 54 (58.1%) of 93 feces in
19 (90.5%) cases within 2 hours. The exceptions were case 10 where a
confirmation test was necessary to detect the eaeA gene of EHEC O26 and case 11 where astA-positive E. coli was
detected on the third run. In the first run, DNA samples extracted from 5 feces
(1, 3, 4, or 7 feces in 6 cases) of symptomatic patients were used and the
causative pathogens were detected from 1 to 5 samples: 1 (in 8 cases: 1, 2, 4,
7, 8, 15, 19, and 21), 2 (in 3 cases: 9, 13, and 20), 3 (in 3 cases: 16, 18,
and 21), 4 (in 3 cases: 5, 6, and 17), and 5 samples (in 3 cases: 3, 12, and
14). Then the causative pathogens were later isolated in a routine laboratory. In
cases 11 and 12, C. perfringens or C. jejuni was detected by duplex SG-qPCR
with more than 105 CFU/g feces from only 1 sample and C. perfringens was then also isolated
from only 1 of 46 samples and C. jejuni from only 1 of 16 samples by culture method. Therefore, the infections with
both these pathogens were determined to be sporadic cases and they were
immediately eliminated as causative pathogens in cases 11 and 12. It was
confirmed that duplex SG-PCR analysis of 5 feces collected from symptomatic
patients was ultimately the most effective screening method for foodborne
pathogens in foodborne outbreaks [10, 13].Duplex SG-PCR
rapidly and accurately demonstrated that 12 (57.1%) of 21 cases were caused
with a single foodborne pathogen such asC. jejuni (6 cases), C. perfringens (3
cases), B. cereus (2 cases), and TDH-producing V. parahaemolyticus (one case). There were also 7 (33.3%) cases
with plural foodborne bacterial pathogens (such as astA-positive E. coli, EPEC, C. jejuni, C. perfringens, A. hydrophila, and P. shigelloides) and 2 (9.5%)
cases with foodborne bacterial pathogens (astA-positive E. coli or EHEC O:26) and norovirus. In
cases 2 and 10, although detection of norovirus is out of the scope of our work,
norovirus and foodborne bacterial pathogens were concomitantly detected by
conventional PCR analysis in our virological laboratory. In case 2 in which
norovirus was detected in 6 of 7 feces, the astA gene of EAEC was detected from 7 of
10 feces and then astA-positive E. coli strains were isolated from 6
samples. In case 10 in which norovirus was detected from 20 of 22 feces, the eae gene of EPEC or EHEC was detected
from 8 of 22 feces and EHEC O26 strains were isolated from 8 of 22 feces. In 7
cases (cases 1, 11, 12, 13, 16, 20, and 21), the pathogenic E. coli strains belonging to astA-positive E. coli and/or EPEC were concomitantly detected with other foodborne bacterial
pathogens. In case 1, the eae gene of
EPEC or EHEC was detected from 4 of 22 feces and the astA gene of EAEC was detected in 3 other feces.  However, duplex SG-PCR could not detect other
virulent genes, including the stx1
and stx2 genes of EHEC. Then EPEC
strains were later isolated from 5 feces and astA-positive E. coli from 4 other feces. In case 12, the astA gene of EAEC was detected in all 5 feces and the eae gene of EPEC or EHEC in 2 feces, but duplex SG-PCR could not detect
other E. coli virulent genes. The
subsequent bacteriological examination could not isolate pathogenic E. coli among nonpathogenic E. coli flora.  In case 16, the C. jejuni specific gene was detected in 6 of 9 feces and the astA gene of EAEC was detected in 5
feces (both genes from 3 feces). C.
jejuni strains were then isolated from 9 of 14 feces, but we were not able
to isolate the pathogenic E. coli strain among nonpathogenic E. coli flora.  In cases 19 to 21 analyzed
improved real-time PCR using 8 primers for the detection of 7 main foodborne
bacteria and astA-positive E. coli, C. jejuni, EPEC, or astA-positive E. coli
were detected
from 1 to 3 fecal samples on the first run and the absence of the other main
foodborne bacteria in the analyzed samples was readily confirmed. In case 20,
the eae gene of EPEC or EHEC was
detected from 2 of 5 fecal samples on the first run and the gyrB gene of P. shigelloides
was
detected separately from other 2 fecal samples on the second run. Then P. shigelloides strains were isolated
from 2 feces, but isolation of the EPEC strain was very difficult due to the
presence of large nonpathogenic E. coli flora in the feces.In almost all
cases, the duplex SG-PCR assay first run detected these causative agents from more
than one of the five feces. Then, in almost all cases, the presence of a
causative agent (presumed from duplex SG-PCR assay) was confirmed by the
results of the final SG-PCR assay run and the bacteriological cultivation of
additional feces. These findings confirmed that for foodborne outbreaks duplex
SG-PCR is a useful tool for the rapid detection of both single and multiple
pathogens.


## 3.3. Quantification of the Causative Agent in 14 Foodborne
Outbreak Cases

Figure2 shows the relationship
between CFU and DNA copy of foodborne pathogens using SG-quantitative PCR
(qPCR) assay in 71 feces from 14 cases examined by viable cell counting.  There was no correlation (r2=0.1183) between CFU and DNA copy of foodborne pathogens in feces, although
almost all pathogens were detected by SG-PCR from feces registering more than
103 CFU/g by viable cell counting. By using SG- qPCR assay
combined with DNA extraction using the QIAamp DNA Stool Mini kit, Bibbal et
al. [28] reported a significant correlation between CFU and DNA copy of ampicillin-resistant Enterobacteriaceae in swine feces. Fu et
al. [29] reported a significant correlation
between CFU and DNA copy of Lactobacillus and total anaerobic bacteria in dog feces but found no correlation between CFU
and DNA copy of C. perfringens. Although accurate quantifications of foodborne
pathogens, including C. jejuni and C. perfringens, in feces were not
completely performed by SG-qPCR in this study, the presence of any foodborne
pathogens at more than 103 CFU/g feces was certainly confirmed by
melting curve analysis. There are two major problems for these
differences. One cause is different sample preparation that was used for CFU
from the feces stored in the transport medium and for qPCR using the mass sample collected for virological
inspection. Another cause is the approach used to construct the standard
curves that were prepared from pure bacterial cultures. These curves do not
relate with the “real” situation of a bacterial quantification in a
faecal sample and can in part explain the absence of correlation between CFU
and DNA copy of foodborne pathogens in faeces.In our routine bacteriological diagnostic laboratory, we used duplex SYBR
Green I PCR assay combined with DNA extraction via QIAamp DNA Stool Mini kit
for the detection of foodborne bacteria from 21 foodborne outbreak cases. The
causative bacteria were detected in almost all cases in 2 hours or less. The
first run was for the detection of 8 main foodborne bacteria and the second run
was for the detection of other unusual suspect bacteria. The results proved
that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, duplex
SG-PCR assay is not only very useful, but is also economically viable for one-step
differentiation of causative bacteria in fecal specimens obtained from
symptomatic patients. This then allows for effective diagnosis and
management of foodborne outbreak.



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*Source: 917623-2009-06-24.xml*