Eur Food Res Technol (2010) 231:985–998

DOI 10.1007/s00217-010-1353-0

ORIGINAL PAPER

A method based on the ligation detection reaction–universal array (LDR–UA) for the detection and characterization of Listeria and Campylobacter strains

Andrea Lauri · Bianca Castiglioni · Marco Severgnini · Chiara Gorni · Paola Mariani

Received: 10 June 2010 / Revised: 4 August 2010 / Accepted: 12 August 2010 / Published online: 1 September 2010© Springer-Verlag 2010

Abstract Listeria and Campylobacter genera includesome of the most widely spread human pathogens acrossEurope and represent a serious health threat, especially tochildren, immunocompromised people and pregnantwomen. Both genera are frequently isolated from farm ani-mals and food; therefore, their rapid detection is importantfor food safety and to prevent disease outbreaks. A rapiddetection approach based on the combination of ligationdetection reaction and universal array (LDR–UA) wasdeveloped to reveal the presence of Listeria and Campylo-bacter pathogenic species and to identify the Division (I, IIand III) of L. monocytogenes isolates. The approach wastested Wrst on reference strains then on Weld isolates. TheLDR–UA approach showed high sensitivity and high speci-Wcity in reliably discriminate target sequences diVering inas little as one base pair, thus facilitating the discriminationof closely related strains.

Keywords Food safety · Diagnostics · Ligation detection reaction · Universal array · Listeria · Campylobacter

Introduction

The Listeria genus includes six species of Gram-positivebacilli: L. monocytogenes, which is considered the most

dangerous Listeria species; L. ivanovii and L. seeligeri, thathave been sporadically reported as causes of disease [1, 2];L. innocua, L. grayi and L. welshimeri, that do not result inclinical disease in humans [3]. L. monocytogenes is dividedinto three evolutionarily serotype lines: Division I (sero-types 1/2b, 3b, 4b, 4d, 4e and 7), Division II (serotypes1/2a, 1/2c, 3a, and 3c) and Division III (serotypes 4a and 4c)[4]. Most of the Listeria clinical isolates belong to DivisionI (about 85%) followed by Division II (15%). Listeria Divi-sion I isolates from food are also predominant with respectto Division II. Listeria Division III is rarely isolated fromboth food and clinical cases and has been reported to bepathogenic primarily to animals [5].

Campylobacter is a characteristic spiral-shaped micro-aerophilic Gram-negative bacterium and the genus includes18 species, 11 of which have been associated with humandiseases [6].

The European Food Safety Authority (EFSA) reportedthat in 2005 and 2007 Campylobacteriosis was the mostprevalent zoonotic disease in humans in the EuropeanUnion, with C. jejuni, C. coli, C. upsaliensis and C. lari thepredominant species [7, 8]. Listeria is also an importantcause of livestock-derived infections and in contrast toother pathogens (e.g. Salmonella), seems not to havedecreased in frequency over the last years [7, 9, 10]. Fur-thermore, Listeriosis is the zoonotic disease with the high-est death rate, estimated over 20% [10, 11].

Poultry is considered a major reservoir of both Campylo-bacter and Listeria, but other farm animals, such as diaryand beef cattle and pigs, and the derived products, consti-tute possible sources of infection. Additional sources ofinfection include Wsh products, water, fruits and vegetables[7, 9, 10, 12].

A quick and precise assay to detect contaminations andthe source of human infections would improve food safety

A. Lauri (&) · C. Gorni · P. MarianiPTP, via A. Einstein, 26900 Lodi, Italye-mail: andrea.lauri@tecnoparco.org

B. CastiglioniIBBA-CNR, via A. Einstein, 26900 Lodi, Italy

M. SevergniniITB-CNR, via F.lli Cervi 93, 20090 Segrate, Milano, Italy

123

986 Eur Food Res Technol (2010) 231:985–998

and medical care. Indeed, fast and sensitive molecular tech-niques are increasingly implemented in routine diagnosistogether with traditional methods, which are based on cul-ture and biochemical protocols [13–15].

Among the molecular approaches, TaqMan PCR hasbeen increasingly used for diagnostic tests as it facilitatesthe rapid and accurate detection of pathogens. In contrast totraditional PCR, TaqMan PCR cuts oV unspeciWc ampliW-cations, thanks to Xuorescently labeled probes speciWc tothe ampliWed target DNA [16–18]. However, the simulta-neous detection of multiple diVerent bacterial species andthe discrimination of subspecies is diYcult and expensive.

The use of DNA microarrays oVers a higher throughputwith respect to PCR and facilitates the simultaneous detec-tion of thousands of species simultaneously, as many spe-ciWc probes can be printed as microspots on the arraysurface [19, 20].

Universal Arrays (UA) are microarrays carrying a seriesof 24–25-bp-long DNA sequences, the so-called Zip Codes,instead of speciWc probes. UAs have been successfully usedwith the ligation detection reaction (LDR) to reveal the pres-ence of DNA target sequences and sequence mismatcheswith a single-nucleotide resolution [21]. The LDR–UA tech-nology has been successfully applied to the detection ofmicroorganisms in diVerent environments [22–24].

Among the target DNA sequences used in molecularmethods for the detection of Listeria spp. are iap (present inall Listeria species; [25]) and hlyA (reported in hemolyticspecies only: L. monocytogenes, L. ivanovii, L. seeligeri;[26, 27]). Regarding Campylobacter spp., target DNAsequences for cadF (called oprF in C. lari and C. upsalien-sis; [28, 29]), cdtB [30] and the ribosomal 16S rRNA geneare commonly used. Besides the 16S rRNA gene, ubiqui-tously present in all bacteria [31], the other genes were cho-sen for their relationship with pathogenicity.

In the present study, the LDR–UA approach was used todevelop a test to simultaneously detect and discriminateCampylobacter spp. and Listeria spp. and Divisions. Thismolecular diagnostic test has the Xexibility of the PCRapproach, the throughput of microarrays, together with thehigh discrimination typical of the LDR.

Materials and methods

All reagents used in the procedures reported in this sectionwere purchased at Sigma-Aldrich (St. Louis, MO, USA), ifnot expressly indicated.

Bacteria strains

Bacteria strains are listed in Tables 1 and 2. They wereobtained from the Collection of the Institute Pasteur (Paris,

France, marked with CIP), from the German Collection ofMicroorganisms and Cell Cultures (Braunschweig,Germany, marked with DSM), from the American TypeCulture Collection (MD, USA, marked with ATCC) andfrom the Salmonella Genetic Stock Centre (Calgary, Canada,marked with SGSC). Field isolates, assessed by PCRaccording to the protocol of Persson and colleagues [31],were kindly provided by Istituto ZooproWlattico Sperimen-tale della Lombardia e dell’Emilia Romagna (IZSLER,Lodi, Italy). Bacteria strains were cultivated according tosupplier recommendations. SpeciWcally, L. monocytogeneswas grown on Columbia Agar and the other Listeria specieson Brain Heart Infusion at 37 °C for 24/48 h with shaking.Campylobacter spp. were grown for 24/48 h on SkirrowAgar in microaerophilic conditions crated using GENbagmicroaer (BioMérieux, France).

Choice of the target sequences

The DNA sequences of the chosen genes (hlyA, iap, cadF,cdtB and 16S rRNA gene) were downloaded from publicdatabases for Campylobacter, Listeria, and for other bacte-ria strains containing homologous genes. HlyA, iap, cadFand cdtB DNA sequences were obtained from NCBI dataset(http://www.ncbi.nlm.nih.gov/), whereas the 16S rRNA genesequences for all Campylobacteriaceae were down-loaded from the Ribosomal Database Project (RDP) website (http://rdp.cme.msu.edu/) [32].

In house re-sequencing was performed for all genes withthe primers listed in Table 3. Bacteria templates are listedin Tables 1 and 2.

Probe design

Sequences obtained by in house re-sequencing and frompublic databases were aligned by ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html). ARB software [33]was used to visualize the sequence alignments and to searchfor sequence polymorphisms.

In order to discriminate and avoid cross-reactivity withhighly similar sequences, probes (listed in Table 4) weredesigned to have the discriminating polymorphism(s) atthe 3� extremity of the discriminating probe (DP) (Fig. 1).The common probe (CP) was designed immediately adja-cent to the 3� end of the DP. Variable bases other than thetarget polymorphisms were masked using Inosine (I),which is an artiWcial base able to equally bind to all fournatural DNA bases. Both CP and DP calculated meltingtemperatures ranged between 65 and 67 °C. DP was mod-iWed at the 5� end with a Cy3 Xuorophore, while the CPwas modiWed at the 5� end with a phosphate and extendedat the 3� end with a cZip Code (reported in Table 4 and in[34]).

123

Eur Food Res Technol (2010) 231:985–998 987

Listeria and Campylobacter detection tool outline

The diagnostic test was created using 34 probe sets, 18 forCampylobacter and 16 for Listeria (Table 4).

The identiWcation of Campylobacter species wasachieved through the detection of the 16S rRNA gene withprobes designed using the consensus sequence from 14Campylobacter species (C. lanienae, C. hyointestinalis,C. fetus, C. sputorum, C. graci, C. rectus, C. showae,

C. concisus, C. curvus, C. helveticus, C. upsaliensis, C. lari,C. coli, C. jejuni). These probes speciWcally recognizeCampylobacter but not other members of the Campylobact-eraceae family, such as Arcobacter and Sulfurospirillum(286 sequences considered) (Fig. 1a). The discrimination ofC. coli, C. jejuni, C. upsaliensis and C. lari was achievedwith 4 probe sets per species that discriminate polymor-phisms found within the cdtB and cadF genes (Table 4;Figs. 1b, c).

Table 1 Molecular signatures for each Listeria and Campylobacter strain are reported

Positive and negative calls are deWned as above or below DT and represented with a black or white box, respectively

Ls_h

lyA

1Li

_hly

A2

Lm_h

lyA

3Lm

_hly

A4

LmI_

hlyA

5Lm

II_hl

yA6

LmIII

_hly

A7

List

_iap

1Lg

_iap

2

Li_i

ap3

Ls_i

ap4

Lm_i

ap5

Lm_i

ap6

LmI_

iap7

LmII_

iap8

LmIII

_iap

9

Listeria monocytogenes - 1/2a ATCC BAA-679Listeria monocytogenes – 1/2a DSM 20600Listeria monocytogenes – 1/2a DSM 12464Listeria monocytogenes - 1/2b ATCC 51780Listeria monocytogenes – 1/2b CIP 105449Listeria monocytogenes – 1/2b CIP 55.143Listeria monocytogenes – 1/2c CIP 105448Listeria monocytogenes – 1/2c CIP 103573Listeria monocytogenes – 3a CIP 54.152Listeria monocytogenes – 3b CIP 78.35Listeria monocytogenes – 3c CIP 78.36Listeria monocytogenes – 4a CIP 105457Listeria monocytogenes - 4b ATCC 19115Listeria monocytogenes – 4b CIP 103322Listeria monocytogenes – 4b DSM 15675Listeria monocytogenes – 4c CIP 54.135Listeria monocytogenes – 4d CIP 105458Listeria monocytogenes – 4e CIP 105459Listeria monocytogenes – 7 CIP 78.43Listeria grayi DSM 20596Listeria grayi DSM 20601Listeria innocua ATCC BAA-680Listeria innocua – 6a DSM 20649Listeria ivanovii subsp. ivanovii DSM 20750Listeria ivanovii subsp. londoniensis DSM 12491Listeria seeligeri – 1/2b DSM 20751Listeria welshimeri DSM 15452Listeria welshimeri - 1/2b DSM 20650

Table 2 Molecular Signatures for each Listeria and Campylobacter strain are reported

Positive and negative calls are deWned as above or below DT and represented with a black or white box, respectively

DB number origin Cam

py16

S1C

ampy

16S2

CcC

adF1

CcC

adF2

CcC

dtB

1C

cCdt

B2

CjC

adF1

CjC

adF2

CjC

dtB

1C

jCdt

B2

ClC

adF1

ClC

adF2

ClC

dtB

1C

lCdt

B2

CuC

adF1

CuC

adF2

CuC

dtB

1C

uCdt

B2

C. jejuni CCUG6824 Human fecesC. jejuni CCUG8714 Human fecesC. jejuni CCUG10937 Human fecesC. jejuni CCUG10954 Human fecesC. jejuni CCUG11284 Bovine fecesC. jejuni CCUG25903 ChickenC. coli CCUG10969 Turkey fecesC. coli CCUG11283 Porcine fecesC. lari CCUG23947 Gull cloacal swabC. upsaliensis CCUG14913 Canine feces

Campylobacter 55 Field Isolate MilkCampylobacter 57 Field Isolate MilkCampylobacter 58 Field Isolate Bovine fecesCampylobacter 59 Field Isolate MilkCampylobacter 60 Field Isolate MilkCampylobacter 61 Field Isolate MilkCampylobacter 63 Field Isolate Milk

123

988 Eur Food Res Technol (2010) 231:985–998

The detection of Listeria was based on variations in iapand hlyA. All Listeria species detection was achieved withiap targeting probe sets. List_iap1 was designed to detect L.innocua, L. monocytogenes, L. ivanovii, L. seeligeri and L.welshimeri, and iap2 to detect L. grayii.

Further iap probe sets were designed to discriminate theListeria pathogenic species: L. ivanovii (Li_iap3), L. seeligeri(Ls_iap4) and L. monocytogenes (Lm_iap5 and Lm_iap6).The three L. monocytogenes subspecies Divisions were alsodiscerned with Lm_iap7 (Division I), Lm_iap8 (Division II)and Lm_iap9 (Division III) (Table 4 and Fig. 1d).

The same probe design scheme was used with hlyA as atarget sequence. Ls_hlyA1 and Li_hlyA2 were designed tospeciWcally recognize L. ivanovii and L. seeligeri, respec-tively. All L. monocytogenes serotypes were detected withLm_hlyA3 and Lm_hlyA4, whereas the Division attribu-tion relied on Lm_hlyA5 (Division I), Lm_hlyA6 (DivisionII) and Lm_hlyA7 (Division III) (Table 4 and Fig. 1e).

In total, with regard to the two target genes iap and hlyA,the presence of L. ivanovii and L. seeligeri was revealed bytwo speciWc probe sets each, whereas the predominantL. monocytogenes was detected by four probe sets and sixadditional probe sets classiWed the strain under investiga-tion in one of the Divisions.

As it is evident from the tool outline, the system wasconceived to provide redundant information. The detectionof a strain was therefore conWrmed at multiple levels:genus, species and, for L. monocytogenes, subspecies. Foreach level, more probe sets were designed providing identi-cal information. This “vertical” and “horizontal” redun-dancy was introduced to confer robustness and versatility tothe system (see “Discussion”).

Polymerase chain reaction (PCR)

Each PCR was performed in a separate tube with 20 ng ofgenomic bacterial DNA (extraction with Gentra kit,

QIAGEN, Hilden, Germany). The primers used are listed inTable 3. PCR conditions were as follows: 4 mM MgCl2, 1UTaq DNA Polymerase (Invitrogen, Carlsbad, California,USA), 1£ reaction buVer, 0.2 �M of each primer, 0.2 mMof each dNTP in a total volume of 20 �L. PCR mix was Wrstdenatured 5� at 94 °C, then cycles of 1� at 94 °C, 1� at 56 °Cand 1� at 72 °C were repeated 40 times. The reaction endedwith 10� at 72 °C extension. Ten microliters of PCR prod-uct was loaded onto a 2% EtBr agarose gel to check thepresence and quality of the ampliWcation product. PCRproducts were puriWed by GenElute PCR Clean-Up Kit(Sigma, Steinheim, Germany), eluted in 50 �L of water andquantiWed with a NanoDrop spectrophotometer.

Universal array (UA) preparation

Phenylen-diisothiocyanate (PDITC) activated chitosanglass slides were used as surfaces [35]. Microarrays wereprepared as described [36], using a MicroGrid II Compact(Biorobotics, UK) contact pin spotter, equipped with Sili-con Microarray pins (Parallel Synthesis Technologies,Santa Clara, CA, USA). Thirty-six Zip Codes wereassigned for the recognition of the target DNA sites. Zip 66and Zip 63 were used as hybridization control and ligationcontrol, respectively. Eight subarrays per slide were posi-tioned such to be hybridized independently with a multi-chamber hybridization system. Each subarray containedfour replicates of each Zip Code distributed according tothe deposition scheme previously reported [36].

Ligation detection reaction (LDR) and universal array hybridization

The LDR was carried out in a volume of 20 �L. The reactionmix included the following: 1 unit of Pfu Ligase (Stratagene,La Jolla, California, USA), 2 �L of 10£ buVer, 50 ng ofeach puriWed PCR product and CP and DP probes to the

Table 3 Primers used in this study

Gene Primer name

Primer sequence Literature

Listeria

iap LisF ATGAATATGAAAAAAGCAACKATC Volokhov et al [20]

LisR ACATARATIGAAGAAGAAGAWARATTATTCCA

hlyA IsoF GTTAATGAACCTACAAGHCCTTCC Volokhov et al [20]

IsoR AACCGTTCTCCACCATTCCCA

Campylobacter

cdtB AL114 GGCACTTGGAATTTGCAAGGCTCAT This study

AL117 GCATCATTTCCTATGCGAATTCCTATAATAGGTC

cadF AL119 GATAACAATGTAAAATTTGAAATCACTCC This study

AL120 AATTTGATCTCTAGTTTCAAGTCTTAAAGC

16S 16S-F GGAGGCAGCAGTAGGGAATA Persson et al. [31]

16S-R TGACGGGCGGTGAGTACAAG

123

Eur Food Res Technol (2010) 231:985–998 989

Tab

le4

Prob

e se

ts u

sed

in th

is s

tudy

: dis

crim

inat

ing

prob

e (D

P) a

nd c

omm

on p

robe

(C

P)

Pro

be s

et

nam

eT

arge

t ge

neD

etec

ted

spec

ies

Zip

co

dePr

obe

type

Sequ

ence

Ls_

hlyA

1hl

yAL

iste

ria

seel

iger

iZ

IP5

DP

CT

CT

AG

CT

CG

CA

TA

GT

AA

CA

AA

GT

TA

AA

AC

TG

CT

TT

CG

AG

GC

G

CP

GC

GA

TG

AG

TG

GC

AA

AT

CA

GT

GA

AA

GG

GG

AT

GT

AG

AA

TT

AA

C

Li_

hlyA

2hl

yAL

iste

ria

ivan

ovii

ZIP

1D

PC

TT

CT

AA

GG

CT

TA

CT

CG

GA

TG

GA

AA

AA

TT

AA

CC

TA

GA

TC

AT

TC

C

CP

GG

TG

CC

TA

TG

TT

GC

GA

GA

TT

CA

AT

GT

TA

CT

TG

GG

AT

GA

AG

Lm

_hly

A3

hlyA

Lis

teri

a m

onoc

ytog

ens

ZIP

41D

PC

TG

TC

TC

AG

GT

GA

TG

TA

GA

AC

TIA

CA

AA

TA

TC

AT

CA

AA

AA

TT

CT

TC

CT

TC

CP

AA

AG

CC

GT

AA

TT

TA

CG

GIG

GC

TC

CG

CA

AA

AG

AT

GA

AG

TT

CA

AA

TC

Lm

_hly

A4

hlyA

Lis

teri

a m

onoc

ytog

ens

ZIP

7D

PG

GG

AT

GA

AA

TA

AA

TT

AT

GA

TC

CT

GA

AG

GT

AA

CG

AA

AT

TG

TT

CA

AC

AT

AA

AA

AC

CP

TG

GA

GC

GA

AA

AC

AA

TA

AA

AG

CA

AG

ITA

GC

TC

AT

TT

CA

CA

TC

GT

CC

Lm

I_hl

yA5

hlyA

Lis

teri

a m

onoc

ytog

ens

I (1

/2b,

3b,

4b,

4d,

4e,

7)

ZIP

33D

PC

AA

CT

CA

GA

AT

AT

AT

TG

AA

AC

AA

CT

TC

AA

AA

GC

TT

AT

AC

AG

AT

GG

AA

AA

AT

C

CP

AA

CA

TC

GA

TC

AC

TC

TG

GA

GG

AT

AC

GT

TG

CT

CA

AT

TC

AA

CA

TC

TC

Lm

II_h

lyA

6hl

yAL

iste

ria

mon

ocyt

ogen

s II

(1

/2a,

1/2

c, 3

a, 3

c)Z

IP21

DP

CG

GC

AA

CC

TC

GG

AG

AC

TT

AC

GIG

AT

AT

TT

TG

AA

AA

AA

GG

C

CP

GC

TA

CT

TT

TA

AT

CG

AG

AA

AC

AC

CA

GG

AG

TT

CC

CA

TT

GC

TT

AT

AC

AA

C

Lm

III_

hlyA

7hl

yAL

iste

ria

mon

ocyt

ogen

s II

I (4

a, 4

b, 4

c)Z

IP17

DP

GC

AG

AA

AA

TC

CT

CC

TG

CA

TA

TA

TC

TC

AA

GT

GT

GG

CA

TA

C

CP

GG

ICG

TC

AA

GT

TT

AT

TT

GA

AA

TT

AT

CG

AC

TA

AT

TC

CC

AT

AG

CA

C

Lis

t_ia

p1ia

pL

iste

ria

mon

ocyt

ogen

eZ

IP9

DP

GA

TT

GC

GG

TIA

CA

GC

AT

TT

GC

TG

CIC

CA

AC

IAT

CG

CA

T

Lis

teri

a iv

anov

iiC

PC

CG

CA

AG

CA

CIG

TA

GT

AG

TIG

AA

GC

TG

GIG

AT

AC

TC

TT

T

Lis

teri

a in

nocu

a

Lis

teri

a se

elig

eri

Lis

teri

a w

elsh

imer

i

Lg_

iap2

iap

Lis

teri

a gr

ayii

ZIP

24D

PG

CT

GG

AA

TA

GC

TG

TC

AC

AG

CA

TT

CG

CT

GC

TC

CA

T

CP

CG

GT

TG

TC

TC

AG

CA

AA

TA

CA

GT

GG

TT

GT

CG

CA

TC

TG

Li_

iap3

iap

Lis

teri

a iv

anov

iiZ

IP6

DP

CC

CC

TG

CA

CC

AG

AA

GT

AA

AA

GA

AG

AA

AC

AA

CT

AC

TC

AA

GC

CP

GC

AA

GC

GG

CA

CC

TG

CT

GC

TG

AA

AC

GA

AA

CC

A

Ls_

iap4

iap

Lis

teri

a se

elig

eri

ZIP

10D

PG

CA

AA

TG

GC

TG

GA

AC

AA

AA

TC

AC

TT

AT

GG

TG

AA

GG

AA

AA

AC

AG

GC

CP

TA

CG

TT

AA

TG

GC

AA

AT

AC

TT

AG

GT

AA

TG

CT

GT

AA

CA

AG

TG

CT

CC

GT

C

Lm

_iap

5ia

pL

iste

ria

mon

ocyt

ogen

sZ

IP36

DP

CG

CA

AC

TT

GG

TT

AA

AC

GT

ICG

TA

GT

GG

CG

CT

GG

T

CP

GT

TG

AT

AA

CA

GT

AT

TA

TT

AC

GT

CIA

TC

AA

AG

GC

GG

AA

CA

AA

AG

TA

AC

TG

Lm

_iap

6ia

pL

iste

ria

mon

ocyt

ogen

sZ

IP35

DP

CT

GT

TG

CA

CC

AA

CA

CA

AG

AA

GT

GA

AA

AA

AG

AA

AC

TA

CT

AIT

CA

AC

AA

GC

CP

TG

CA

CC

TG

CT

GC

AG

AA

AC

AA

AA

AC

TG

AA

GT

AA

AA

CA

AA

CT

AC

AC

AA

GC

Lm

I_ia

p7ia

pL

iste

ria

mon

ocyt

ogen

s I

(1/2

b, 3

b, 4

b, 4

d, 4

e, 7

)Z

IP39

DP

CG

TT

CG

TA

GT

GG

CG

CT

GG

TG

TT

GA

TA

AC

AG

TA

TT

AT

TA

CG

TC

A

CP

AT

CA

AA

GG

CG

GA

AC

AA

AA

GT

AA

CT

GT

TG

AA

TC

AA

CC

GA

AT

CT

AA

TG

GC

Lm

II_i

ap8

iap

Lis

teri

a m

onoc

ytog

ens

II

(1/2

a, 1

/2c,

3a,

3c)

ZIP

5 BD

PC

GA

TG

GA

AA

AA

CT

GG

TT

TC

GT

TA

AC

GG

TA

AA

TA

CT

TA

AC

TG

AC

AA

AG

CA

GT

CP

AA

GC

AC

TC

CA

GT

TG

CA

CC

AA

CA

CA

AG

AA

GT

GA

AA

AA

AG

AA

AC

TA

CT

AC

123

990 Eur Food Res Technol (2010) 231:985–998

Tab

le4

cont

inue

d

Pro

be s

et

nam

eT

arge

t ge

neD

etec

ted

spec

ies

Zip

cod

ePr

obe

type

Seq

uenc

e

Lm

III_

iap9

iap

Lis

teri

a m

onoc

ytog

ens

III

(4a,

4b,

4c)

ZIP

12D

PG

TA

CC

GG

GT

CA

AA

AA

TT

AC

AA

GT

GA

CT

GA

AG

TA

AA

TG

AG

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Eur Food Res Technol (2010) 231:985–998 991

Wnal concentration of 50 nM each. The oligo-mix alsocontained a discriminating probe and a common probe spe-ciWc for the synthetic oligonucleotide used as ligation control(5�-AGC CGC GAA CAC CAC GAT CGA CCG GCGCGC GCA GCT GCA GCT TGC TCA TG-3�). The mix wasWrst denatured for 10� at 94 °C, followed by 30� at 94 °C and4� at 65 °C, repeated for 30 cycles. Then, 45 �L of hybridiza-tion buVer (1.10 M NaCl, 0.11 M sodium citrate, 7 �gsalmon sperm DNA) was added to the reaction, denatured at94 °C for 3� and immediately hybridized on UA at 65 °C for2 h. The UA was then washed at 65 °C in SSC 2£ (0.3 MNaCl, 0.03 M Sodium Citrate) and 0.1% SDS for 5�, in SSC2£ for 5�, in SSC 1£ for 5� and in SSC 0.5£ for 5�.

Array scanning, data acquisition and processing

Hybridized UAs were scanned by the laser scanner ScanAr-ray LITE (Perkin Elmer, Waltham, Massachusetts, USA).Images were acquired avoiding saturation and then ana-lyzed with the QuantArray software (Packard BioChipTechnologies, Billerica, USA) to quantify the signal/noiseratio (SNR). SNR is deWned as the ratio of the spot meanintensity to the Xuorescence from an area immediately out-side that represents the local background. For each experi-ment, the median SNR value of the four spot replicates ofeach Zip Code (and therefore of each probe set) was con-sidered. The median value is here referred to as speciWcSNR or unspeciWc SNR, depending on whether it was regis-tered in the presence of the probe set–speciWc target or onlyof the closest unspeciWc targets, respectively. For speciWcSNRs, at least 2 experiments were carried out per probe set.

The mean of unspeciWc SNRs (at least from 3 experi-ments) plus 3 times their standard deviation (SD) deWnedthe detection threshold (DT). For instance, to deWne the DTof probe sets targeting L. monocytogenes I (LmI_hlyA5 andLmI_iap7), experiments were carried out on the closestunspeciWc target (L. monocytogenes II and III). For theother Listeria and Campylobacter species, experimentswere performed on the other species belonging to the samegenus and, for Campylobacter 16S rRNA gene targetingprobes (Campy16S1 and Campy16S2), with no DNA.

Assuming the unspeciWc SNR values follow a NormalDistribution, only 0.1% of these values passed the DT.Therefore, values above this threshold are distinct frombackground with 99.9% conWdence.

Results

Implementation of the tool on type strains

The LDR–UA-based assay was tested on PCR productsobtained from genomic DNA extracted from theT

able

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AA

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CC

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CA

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AA

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AG

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GT

TT

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CA

AA

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AT

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CP

AC

AG

GT

AG

AA

TG

GT

GC

AG

CC

CG

GIG

GC

123

992 Eur Food Res Technol (2010) 231:985–998

Campylobacter and Listeria type strains reported inTables 1 and 2 and with the primers reported in Table 3.The LDR was performed with a total of 34 probe sets, com-prising 18 probe pairs for Campylobacter and 16 for Listeria(Table 4 and Fig. 1).

SNR unspeciWc values never exceeded twice the meanvalue and the calculated standard deviations (SD) werenever greater than 50% of the mean value. The DT wasestablished for each probe set (see “Materials and Meth-ods”). DT values resulted to be pretty variable both for Lis-teria and for Campylobacter targeting probe sets, andranged from 19.01 (List_iap1) to 106.25 (LmI_hlyA5).Similarly, the mean of speciWc SNR values ranged from3970.25 (Campy16S2) to 77.40 (Li_iap3) and were gener-ally higher for the Campylobacter targeting probes.

The distribution of unspeciWc SNR values and of speciWcSNR values relative to each probe set resulted to be alwayswell distinct, and no ambiguous results were observed, as con-Wrmed by t test analyses (P < 0.001). Furthermore, speciWcSNR values resulted to be always greater than the backgroundsignal mean plus 4£SD, therefore well above the DT, as only0.003% of background values passed this limit (Figs. 2 and 3).

The LDR–UA experiment results can be schematizedwith a positive or negative signal for each probe set, whenits SNR value was above or below the DT, respectively.Taken together, the positive and negative signals deWnedthe hybridization pattern of each strain, i.e. its molecularsignature (Tables 1 and 2). All the investigated strainsshowed a molecular signature identical to that expectedfrom the sequence data.

Fig. 1 For each probe set, the 10 bp at the 3� end of DP and the 10 bp at the 5� end of CP are re-ported and highlighted with a diVerent shadowing. These por-tions are aligned with speciWc targets, enclosed in an open box, and with the consensus of homologous region from other species. Identities are high-lighted with dots and gaps with dashes. Similarities among strains, as determined based on whole gene sequence, are under-lined with dendrograms. a 16S rRNA gene; “Related species” indicates species of the Arcobacter and Sulfurospirillum genera. b cdtB. c cadF. d iap. e hlyA

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Eur Food Res Technol (2010) 231:985–998 993

Fig. 1 continued

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994 Eur Food Res Technol (2010) 231:985–998

The List_iap1 probe set detected all Listeria strains butL. grayii that was speciWcally recognized by Lg_iap2(Fig. 1d). The Listeria species virulent to humans (L. seeli-geri, L. ivanovii and L. monocytogenes) were correctlydetected by the speciWc probe sets (Ls_hlyA1 + Ls_iap4,Li_hlyA2 + Li_iap3 and Lm_hlyA3 + Lm_hlyA4 + Lm_iap5 + Lm_iap6, respectively). The Division assignment ofL. monocytogenes strains was achieved with theLmI_hlyA5, LmII_hlyA6, LmIII_hlyA7, LmI_iap7,LmII_iap8, LmIII_iap9 probe sets and corresponded to thatexpected from the serotype (Figs. 1d, e, Table 4). From lit-erature data, L. monocytogenes 4b strains were predomi-

nantly assigned to Divisions I and with less frequency toDivision III [37]. The L. monocytogenes 4b strains investi-gated in this study (ATCC 19115, CIP 103322, DSM15675) could be clearly assigned to Division I.

All Campylobacter species were recognized by theprobe sets targeting the 16S rRNA gene (Campy16S1 andCampy16S2) (Fig. 1a). The four Campylobacter speciesreacted as expected with the speciWc probe sets: C. jejuniwith CjCadF1 CjCadF2 CjCdtB1 CjCdtB2, C. coli withCcCadF1 CcCadF2 CcCdtB1 CcCdtB2, C. lari with ClCadF1ClCadF2 ClCdtB1 ClCdtB2 and C. upsaliensis withCuCadF1 CuCadF2 CuCdtB1 CuCdtB2 (Fig. 1b, c).

Fig. 2 Mean of unspeciWc and speciWc SNR values and relative standard deviations are indicated for each probe set for Campylo-bacter. The DT level is also re-ported and corresponds to the background mean plus 3£SD. Values are reported in a logarith-mic scale

1

10

100

1000

10000

Campy

16S1

Campy

16S2

CcCad

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B1

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ClCad

F1

ClCad

F2

ClCdt

B1

ClCdt

B2

CuCad

F1

CuCad

F2

CuCdt

B1

CuCdt

B2

SNR-Specific

SNR-Unspecific

DT

Fig. 3 Mean of unspeciWc and speciWc SNR values and relative standard deviations are indicated for each probe set for Listeria. The DT level is also reported and corresponds to the back-ground mean plus 3£SD. Values are reported in a logarithmic scale

1

10

100

1000

10000

Ls_h

lyA1

Li_hly

A2

Lm_h

lyA3

Lm_h

lyA4

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iap7

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iap8

LmIII

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SNR-Specific

SNR-Unspecific

DT

123

Eur Food Res Technol (2010) 231:985–998 995

Implementation of the tool on Weld isolates

The tool was tested on seven Campylobacter Weld isolatesfrom bovine feces and milk. These samples were foundpositive for the presence of C. jejuni by a routine PCR test[31], whereas primers speciWc to C. coli, also included inthe test, did not give positive result (not shown).

LDR–UA tests conducted on these samples conWrmedthe presence of C. jejuni as all were positive for C. jejuni-speciWc probe sets (CjCadF1, CjCadF2, CjCdtB1,CjCdtB2) (Table 2). One sample (Campylobacter 55) wasalso positive for all C. coli-speciWc probe sets (CcCadF1,CcCadF2, CcCdtB1, CcCdtB2), for CuCdtB1, and CuCdtB2,but not CuCadF1 and CuCadF2, speciWcally targetingC. upsaliensis. Positive calls were unequivocally above theDT level, as they exceeded this limit about twofold(CuCdtB1 and CuCdtB2), about Wvefold (CcCadF1 andCcCadF2) and about sixfold (CcCdtB1 and CcCdtB2).

The coexistence of diVerent species in the sampleCampylobacter 55 was conWrmed by sequencing thePCR product used as LDR target. The sequence chro-matogram clearly showed overlapping peaks in corre-spondence to the three species divergent bases, rulingout the possibility of probe cross-reactivity or of unspeciWchybridization.

Only one probe set (Campy16S1) failed to react withthree Campylobacter isolates (Campylobacter 55, 59, 63).However, the assignment of these isolates to this genus wasconWrmed by the second 16S rRNA gene targeting probeset (Campy16S2) that was positive in all samples.

SNR values from negative spots were always lower thanthe unspeciWc mean plus 1£SD and therefore well belowthe DT, set at 3£SD plus the mean.

Discussion

Listeria and Campylobacter are among the most diVusedfoodborne pathogens in Europe. Several are the infectionsources and concern all kinds of food from poultry, pork,beef to diary products. Contaminated water can spread bac-teria into Wsh cultures or vegetable cultivations.

The rapid detection of Campylobacter and Listeriacontamination is therefore important for food safety and toprevent outbreaks. The precise determination of the con-taminating species is also crucial, as both genera includestrains that are not a threat to human health and if notdistinguished from the pathogenic strains can create falsepositive detections and alarm.

To meet the need of a reliable and robust detection ofthese two genera, the LDR–UA technology was success-fully implemented in a new diagnostic tool.

Probe performance

Probe sets used in this study were designed to couple hightarget speciWcity to strain secure detection.

The DT, independently calculated for each probe set,allowed positive/negative calls with 99.9% conWdence.Within each probe set data, the unspeciWc SNR values, deW-ned on the basis of experiments conducted on unspeciWc tar-gets, showed to be contained within a short range (mean plus/minus 50%). On the other hand, diVerences among DTs weremore relevant. This could be due to the variation of the meanvalues rather than of the SD values, and therefore seemed todepend on the probe sequences rather than on random factors.These data conWrmed the need to set DTs independently foreach probe set, so to distinguish real signals from artifacts.

SNRs registered in the presence of speciWc targets wereabove the thresholds and clearly distinct from those consid-ered as background, both in experiments conducted onknown Listeria and Campylobacter strains and in blindtests performed on Campylobacter spp., as the lowest valuewas still higher than mean plus 4£SD. Nevertheless, set-ting the DT at this level is dangerous as the possibility thatspeciWc signals fall below this limit is not negligible. Thisis especially true for those probe sets with a low speciWcSNR mean such as Li_iap3 or ClCadF1 (Figs. 2 and 3).

The 16S targeting probe sets (Campy16S1 andCampy16S2) were experimentally tested on C. jejuni, C.coli, C. lari and C. upsaliensis, even though they could vir-tually detect many other Campylobacter spp., as the probetarget sequence is conserved. Only the detection of C. rec-tus, a species possibly related to dental disease [38], mightbe diYcult because of variations in probe portions particu-larly sensitive to mismatches, i.e. the 5 bp at the 3� end ofDP and the 5 bp at the 5� end of CP [39]. SpeciWcally, C.rectus presents mismatches in the last base at the 5� of theCampy16S1 CP and in the second last position from the 3�

end of the Campy16S2 DP. Similarly, the Campy16S2probe set might not work on all C. fetus strains, as the C inthe third position from the 5� end of the CP was found to bereplaced by a T in 4/30 strains. Nevertheless, in case ofCampy16S2 malfunctioning on C. fetus, the detection ofthis species would be assured by the informatively redun-dant Campy16S1 probe set (Fig. 1a).

Although not experimentally tested, the cross-reaction ofCampy16S1 and Campy16S2 probe sets and any speciesnot belonging to the Campylobacter genus is to be excludedbecause of the poor homology between the probes and thetarget sequences. In fact, the Arcobacter genus that showedthe highest homology to Campylobacter has at least onemismatch positioned at the 3� end of the DP. All the otherspecies have more mismatches in the 5 bp at the 3� end ofDP and at the 5� end of CP and beyond (Fig. 1a).

123

996 Eur Food Res Technol (2010) 231:985–998

Although no probe sets returned ambiguous results, theSNR mean values were pretty variable for both Campylo-bacter and Listeria. DiVerences could not be correlatedwith probe features such as diVerent annealing tempera-tures, GC content, presence of Inosines or predicted sec-ondary structures. An explanation, though, to the diVerentprobe performance could be given by a nonobvious combi-nation of these factors.

LDR–UA tool performance

The aim of the work here described was to develop a diag-nostic tool that can quickly and precisely detect Campylo-bacter and Listeria spp., even in the presence ofconcomitant strains. The LDR–UA technology was chosenfor its great sensitivity and discriminating power thataccompany its customizability and Xexibility. The LDR–UA experiments could be carried out in one working day,thus conWrming its rapidity compared to traditional meth-ods [36, 40].

The implementation of 34 probe sets allowed the detec-tion of all Listeria and Campylobacter strains and the iden-tiWcation of the species pathogenic to humans.

For L. monocytogenes, by far the most dangerous andfrequently isolated in clinical cases among Listeria species,the determination of the Division was also possible. In caseof outbreaks, this would be of great help in L. monocytoge-nes contamination tracking and for epidemiological studies.Moreover, as the three Divisions have separated evolution-ary history, host adaptation and clinical symptoms, theDivision determination could be of great help to understandthe pathogenic potential of an isolate [41]. For L. monocyt-ogenes 4b, the mere determination of the strain serotype,achieved with immunochemical methods, does not indicatethe Division it belongs to, as 4b serotypes are split in Divi-sions I and III. So far, molecular PCR-based tests contrib-uted to the Division determination [42].

The detection of Listeria harmless species, such as L.innocua, that occupy ecological niches similar to L. mono-cytogenes, is of great consequence as it indicates a Listeriafavorable environment, thus the potential presence also ofL. monocytogenes [43].

The possibility to customize the pathogen detectionassay, simply by preparing the speciWc probe mix, gives theLDR–UA system great Xexibility and allows adapting theexperiment outline to any situation, for instance, decidingwhich speciWc pathogens to detect or what resolution isneeded. This oVers a solution to the intrinsic rigidity oftraditional diagnostic microarrays, with probes printeddirectly onto the chip, as in this case the tool customizationrequires the preparation of brand new microarray slideseach time.

The LDR–UA method here outlined has advantages alsoover the TaqMan PCR approach. First, to be equally infor-mative, the TaqMan approach must implement at least 34probes, with clear drawbacks in terms of handiness, as mul-tiplexing in one or few reactions is obviously diYcult. Sec-ondly, with the LDR–UA, a single base mismatch issuYcient to prevent the ligation reaction, conferring to thetool the highest discrimination power of single-nucleotidepolymorphisms (SNPs). The same performance is reachedin TaqMan experiments only with two diVerently markedcompeting probes per SNP, with a consequent costs raise.

The LDR–UA couples the ampliWcation power typical ofPCR to the throughput described for microarrays. In termsof sensitivity, the LDR–UA approach performs better thaneach of these two techniques because it adds, on top of thePCR exponential ampliWcation, the linear ampliWcation ofthe LDR. The hybridization of UA with LDR products per-formed with less that 1 ng of target DNA still gave a clearsignal ([36] and data not shown), thus making this diagnos-tic system suitable for the detection of very small amountsof bacteria. In order to increase reliability, the strain detec-tion was organized in multiple levels: genus, species and,for L. monocytogenes, Divisions. The positive signal fromeach species or Division-speciWc probe set was conWrmedby the signal from probe sets with broader speciWcity. Ateach level, more probe sets targeted diVerent PCR productsrepresenting the same pathogen, thus providing soundinformation about the presence of a speciWc genus, speciesand Division. The redundant detection of one strain confersrobustness to the system and reliability to the output.

The suitability of the tool for routine diagnostics on Weldisolates was conWrmed by the output of a parallel diagnostictest based on PCR (data not shown). Moreover, the LDR–UA assay resulted more sensitive and informative wherethe presence of more Campylobacter species was detected(e.g. isolate Campylobacter 55; Table 2). However, it can-not be ruled out that the routine PCR method failed todetect these additional species because of their low concen-tration in the sample. The absence of positive calls for thecadF targeting probes (CcCadF1 and CcCadF2) might bedue to a too low concentration of the bacteria and the con-sequent impossibility to detect, at this concentration, theless eYciently ampliWable cadF gene. Alternatively, thisstrain might really lack the cadF gene and be not virulent.In a similar fashion, for three strains (Campylobacter 55, 59and 63) only one 16S rRNA gene targeting probe set(Campy16S2) gave signal above background, whereas thesignal from Campy16S1 was below the DT. These casesproved that the choice of implementing more target genesper species/strain and more probe sets for the same targetgene was appropriate, since it allowed the successful detec-tion of species that would have been otherwise missed.

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The next step will be the validation of the tool directlyon Weld samples (food and specimens). The bacteria enrich-ment and the PCR ampliWcation protocols, tested andapplied to the routine PCR-based detection [31] and withthe Campylobacter-speciWc primers used in this study (pre-liminary data not shown), can be easily implemented in theLDR–UA approach.

The LDR–UA technique, here described with its appli-cation to the detection of Campylobacter and Listeria, con-Wrmed high performances. Moreover, with respect to othertechniques (TaqMan, microarray) the LDR–UA can behighly competitive and cost eVective and is a perfect solu-tion for the routine simultaneous detection of 15–40 targets[40].

Acknowledgments We are particularly thankful to Dr. Mario Luiniand Dr. Valentina Benedetti from IZSLER, Lodi, Italy, for providingWeld isolates, for sharing PCR results on Campylobacter Weld isolates,for help in Campylobacter cultivation, for critical reading of the man-uscript and for fruitful discussion.

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