Journal of Microbiological Methods 154 (2018) 107–111

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Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

Rapid detection of Yersinia enterocolitica serotype O:3 using a duplex PCR T

assay
Leonardo Alves Rusaka,b, , Rodrigo de Castro Lisboa Pereirab, Isabelle Geoffroy Freitagb,c,
⁎

Cristina Barroso Hoferc, Ernesto Hoferb, Marise Dutra Asensia, Deyse Christina Vallimb
a
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Pesquisa em Infecção Hospitalar, Rio de Janeiro /RJ, Brazil
b
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Zoonoses Bacterianas/Setor Listeria, Rio de Janeiro /RJ, Brazil
c
Programa de Pós-Graduação em Doenças Infecciosas e Parasitárias, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, /RJ, Brazil

ARTICLE INFO ABSTRACT

Keywords: Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal
Detection diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the
PCR primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and
Screening method identification of Y. enterocolitica from various sources on selective media have been seldom successful due to
Yersinia enterocolitica O:3
several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we
Yersinia spp
developed a single duplex PCR assay for the detection of Y. enterocolitica ssp. palearctica bioserotype 4/O:3 using
DNA extracted from a source. We combined the primer for tufA (elongation factor Tu) with the primer for rfbC
(the biosynthesis of the O side chain) in one single reaction, which showed good results when we analyzed 88
Yersinia strains and when it was tested in the DNA from stool samples of two groups of pregnant women, one
comprising HIV-positive women and the other comprising of HIV-negative women. Furthermore, the duplex PCR
assay was found to be 16 times better in detecting Yersinia spp. in stool samples than the culture-based method.
In addition, it was found to be a rapid screening method for the detection of Y. enterocolitica serotype O:3, and it
could still detect other Y. enterocolitica serotypes and Yersinia species as well. We anticipate that the duplex PCR
assay could be a useful tool for hospital and veterinary surveillance studies on Yersinia worldwide.

1. Introduction global relevance (Batzilla et al., 2011; Fredriksson-Ahomaa et al.,

Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a
zoonotic agent that causes gastrointestinal diseases such as en-
terocolitis, acute mesenteric lymphadenitis (mimicking appendicitis),
and terminal ileitis and extraintestinal disorders such as cellulitis, me-
ningitis, arthritis (postinfection sequelae), and septicemia in humans
(Bottone, 2015; Petsios et al., 2016). Currently, Y. enterocolitica species
are divided into two subspecies, enterocolitica and palearctica, and the
latter subspecies is the one to which the bioserotype 4/O:3 belongs
(Drummond et al., 2012). The bioserotype 4/O:3 is generally isolated
from pigs and humans in Europe, where it is still the primary patho-
genic bioserotype (Fredriksson-Ahomaa et al., 2006a). The other bio-
serotypes that predominate in human and animal illnesses include 1B/
O:8, 2/O:9, and 2/O:5,27 (Bottone, 2015). Y. enterocolitica serotype O:3
biotype 4 has a high public health relevance in Europe, where it com-
prises about 80%–90% of human isolates, along with an increasing
2012).
The culture and identification of pathogenic Y. enterocolitica from
clinical, food, and environmental samples, even on selective media,
have been seldom successful because of the large variety of the colonies
of enterobacterial species, which compete with the low number of Y.
enterocolitica colonies (Fredriksson-Ahomaa and Korkeala, 2003). Tra-
ditional culturing methods have several limitations such as long in-
cubation steps and lack of biochemical identification between species.
All processes, including serological and biochemical analyses, are time-
consuming and generally not available in routine laboratories
(Thoerner et al., 2003; Fredriksson-Ahomaa et al., 2006b). Moreover,
the fact that Y. enterocolitica has the ability to persist in a sample in a
viable, but nonculturable, state could be a big issue for conventional
culture-dependent methods (Alexandrino et al., 2004).
In an attempt to overcome the problems related to traditional cul-
ture-based methods, the direct molecular detection of specific nucleic

⁎
Corresponding author at: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Pesquisa em Infecção Hospitalar, Av. Brasil, n° 4365 – Pavilhão Rocha
Lima - 3°Andar, sala 314 – Manguinhos, Rio de Janeiro/RJ Zip Code: 21040-900, Brazil.
E-mail address: rusak@ioc.fiocruz.br (L.A. Rusak).

https://doi.org/10.1016/j.mimet.2018.10.014
Received 7 September 2018; Received in revised form 21 October 2018; Accepted 22 October 2018
Available online 23 October 2018
0167-7012/ © 2018 Elsevier B.V. All rights reserved.
L.A. Rusak et al. Journal of Microbiological Methods 154 (2018) 107–111

acid targets has been carried out worldwide for the diagnosis of Yersinia Table 1
(Cocolin and Comi, 2005). In this context, PCR is the most used nucleic Listing of strains used in Duplex PCR analyses for tufA and rfbC amplification.
acid amplification technique that can detect a target with high sensi- Species Serotype Source/ N° of Positive for
tivity and specificity (Fredriksson-Ahomaa et al., 2006b). Strain isolates
For the detection of Y. enterocolitica, PCR assays have been devel- tested rfbC TufA
oped to target the primary chromosomal (ail, inv, and yst) and plas-
Yersinia Wild strains
midial (yadA or virF) virulence genes (Cocolin and Comi, 2005; Yersinia enterocolitica O:1,2,3 Animal 1 − +
Fredriksson-Ahomaa et al., 2006b). However, the PCR assays for these Yersinia enterocolitica O:2,3 Animal 1 − +
virulence targets can identify only the strains that harbor these viru- Yersinia enterocolitica O:3 Animal/ 64 + +
lence genes. This could be an issue for detection and identification Human
Yersinia enterocolitica O:4 Human 1 − +
purposes because strains lacking these virulence genes could harbor
Yersinia enterocolitica O:5 Animal/ 4 − +
other virulence genes and have attributes such as production of en- Human
terotoxins, invasion of epithelial cells in vitro, and survival inside Yersinia enterocolitica O:6 Human 1 − +
macrophages, indicating their pathogenic potential (Bhagat and Virdi, Yersinia enterocolitica O:7 Human 2 − +
Yersinia enterocolitica O:8 Animal 1 − +
2011).
Yersinia enterocolitica O:18 Animal 1 − +
To explore a gene that could be used as a marker for the Yersinia Yersinia enterocolitica O:19,8 Animal 1 − +
genus, we selected the tuf (elongation factor Tu) gene. This gene ex- Yersinia enterocolitica O:22 Animal 1 − +
hibited better discriminatory power than the 16S rRNA sequence ana- Yersinia enterocolitica O:47 Animal 1 − +
lysis for identification purposes (Hwang et al., 2011). The tuf gene is Yersinia Type strains
duplicated as tufA and tufB in the majority of enterobacterial genomes Yersinia enterocolitica O:3 CIP 81.41 1 + +
(Isabel et al., 2008). The levels of tufA nucleotide sequence identity Yersinia enterocolitica O:5,27 CIP 106676 1 − +
Yersinia enterocolitica O:6,30 CIP 107202 1 − +
within Yersinia genus have been reported to be 95.9%–96.4%, which
Yersinia enterocolitica O:8 CIP 80.27 T 1 − +
are higher than 90.8%–94.7% of tufB nucleic acid sequence identity Yersinia enterocolitica O:9 CIP 81.42 1 − +
levels. This shows that the tufA gene could be a useful target in diag- Yersinia kristensenii CIP 9993 1 − +
nostic purposes for the identification of Yersinia species (Isabel et al., Yersinia frederiksenii CIP 8029 1 − +
2008). Yersinia ruckerii ATCC 1 − +
29473
On the other hand, regarding the serotype O:3 marker, we selected Yersinia pseudotuberculosis IAL 1791 1 − +
the gene localized in the rfb cluster, which is responsible for the bio-
Negative Control
synthesis of the O side chain of Y. enterocolitica serotype O:3 (Weynants
Escherichia coli ATCC 1 − −
et al., 1996). Within the rfb cluster, Weynants et al. (1996) designed a 25922
primer for the rfbC gene, which could be used for the specific detection Salmonella enterica subsp. ATCC 1 − −
of Y. enterocolitica serotype O:3 from fecal samples. enterica 10708
We aimed to develop a single duplex PCR assay for the detection Citrobacter freundii ATCC 8090 1 − −
Enterobacter sakazakii ATCC 1 − −
and identification of Y. enterocolita bioserotype 4/O:3, and also to de- 29004
tect other members of Yersinia genus, in a rapid, inexpensive, and less Proteus vulgaris ATCC 1 − −
laborious manner compared with methods that use sequencing. To 29513
develop the duplex PCR assay, we combined the primer for the tufA
gene as a Yersinia genus marker with the primer for the rfbC gene as a (−) - negative; (+) - positive; CIP - Collection de l'Institut Pasteur; ATCC -
American Type Culture Collection; IAL - Instituto Adolfo Lutz.
serotype O:3 marker, which is exclusive to Y. enterocolitica (Isabel et al.,
2008; Paradis et al., 2005; Weynants et al., 1996).
2.2. Duplex PCR assay and cycling conditions

2. Materials and methods

The duplex PCR assay was performed for all strains (Table 1). The
genomic DNA of all strains was extracted using a DNA extraction kit
2.1. Bacterial strains
(DNeasy Blood & Tissue Kit; Qiagen, Hilden, Germany), following the
manufacturer's guidelines.
A total of 79 Yersinia wild strains were selected to evaluate the
The duplex PCR comprised the following primer pairs: for the tufA
duplex PCR assay. These strains were composed of 15 Y. enterocolitica
gene forward (5′-ACATCCTGTTGGGTCGYCA-3′) and reverse (5′-TCTT
serotype O:3 strains from human sources, all of them kindly provided
TGCTCAGAATATAAACTTCTGA-3′) (Dalmasso et al., 2014), as a Yer-
by the collection of the Brazilian Reference Center on Yersinia spp.,
sinia genus marker, to amplify a 587-bp fragment of tufA gene. The
other than Y. pestis, and 64 Y. enterocolitica strains of various serotypes
primer pair for the rfbC gene was forward (5′-CGCATCTGGGACACTA
from human and animal sources. Nine Yersinia-type strains were used as
ATTCG-3′) and reverse (5′-CCACGAATTCCATCAAAACCACC-3′)
positive control for the PCR assay, comprising five Y. enterocolitica
(Weynants et al., 1996), which amplifies a 405-bp fragment of rfbC and
strains of several serotypes and four Yersinia strains of other species,
was used as serotype O:3 marker (Fig. 1).
including Y. kristensenii, Y. frederiksenii, Y. ruckeri, and Y. pseudotu-
The reaction was carried out using Platinum PCR Super Mix Kit
berculosis. The following five strains were used as negative controls:
(Invitrogen, Carlsbad, USA) in a final volume of 50 μl, containing 5×
Escherichia coli, Salmonella enterica ssp. enterica, Citrobacter freundii,
PCR buffer, 25 mM MgCl2, 2 mM dNTP mix, 0.5 μl of 25 pmol of each
Enterobacter sakazakii, and Proteus vulgaris (Table 1).
primer, 5 U/μl Taq DNA polymerase, and 2 μl of template DNA. The
These wild strains, the Yersinia-type strains, and the negative con-
PCR cycling conditions were as follows: an initial denaturation step of
trol strains belonged to the Listeria collection CLIST (Laboratory of
5 min at 94 °C, followed by 35 cycles, each consisting of denaturation
Bacterial Zoonosis at Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro,
for 1 min at 94 °C, annealing for 1 min at 56 °C, extension for 1 min at
Brazil).
72 °C, and a final extension step of 5 min at 72 °C.
All the wild strains used in the duplex PCR assay were previously
The PCR product was run on a 1% agarose gel (Sigma-Aldrich, Saint
bioserotyped by the abovementioned reference laboratories (Rusak
Louis, MO, USA) at a constant voltage of 100 V for 60 min in a 0.5×
et al., 2014).
TBE (Tris/boric acid/EDTA) (Bio-Rad, Hercules, CA, USA) buffer. The

108
L.A. Rusak et al.
Fig. 1. Duplex PCR assay detects the rfbC (405 bp) and the tufA (587pb) genes.
Negative controls lanes 2 to 6; Yersinia genus controls lanes 7 to 10; Yersinia
enterocolitica serotype O:3 controls lanes 11 to 12; Lane 1–100-bp DNA marker
(Invitrogen, Carlsbad, USA); lane 2 - Escherichia coli ATCC 25922; lane 3-
Salmonella enterica subsp. enterica ATCC 10708; lane 4 - Citrobacter freundii
ATCC 8090; lane 5 - Proteus vulgaris ATCC 29513; lane 6 – Enterobacter sakazakii
ATCC 29004; Lane 7 - Yersinia kristensenii CIP 9993; Lane 8 - Yersinia freder-
iksenii CIP 8029; Lane 9 - Yersinia ruckerii ATCC 29473; Lane 10 - Yersinia
pseudotuberculosis IAL 1793; Lane 11 - Yersinia enterocolitica serotype O:3
(human wild strain); Lane 12- Yersinia enterocolitica serotype O:3 CIP 81.41;
Lane 13 - reagent blank.
gel was stained with ethidium bromide (Bio-Rad), and the PCR product
was visualized under UV light.
2.3. Sequencing of the tufA and rfbC amplified fragments and BLASTN
search
The strain type CIP 81.14 was chosen for the amplification of its
tufA and rfbC fragments. Both the primer pairs for tufA and rfbC and the
cycling conditions have been mentioned above. The PDTIS-IOC DNA
Sequencing Platform was used to perform the sequencing of the am-
plified DNA fragments of these two genes using Big Dye Terminator
v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA),
after which they were analyzed using an ABI Prism 3100 genetic ana-
lyzer (Applied Biosystems). We then performed a BLASTN search using
the sequenced fragments in all Yersinia species tufA genes and in all
related tufA genes out of the Yersinia genus in the NCBI database. The
sequences with high levels of similarity with those of CIP 81.14 were
aligned with the sequences of tufA and rfbC primers.
2.4. Evaluating the detection of Y. enterocolitica serotype O:3/Yersinia spp.
through the duplex PCR assay and culture-based method using stool samples
This study was performed during 2015–2016 with the primary
purpose of testing the capability of the duplex PCR assay to detect Y.
enterocolitica serotype O:3/Yersinia spp. directly from stool sample DNA
and comparing it with the isolation of Y. enterocolitica serotype O:3/
Yersinia spp. from stool sample using the culture method.
Two groups of patients were evaluated using the assay, one was
composed of healthy pregnant women from Petrópolis-RJ and other
was composed of HIV-positive pregnant women from Instituto de
Puericultura e Pediatria Martagão Gesteira (Federal University of Rio
de Janeiro, Rio de Janeiro, Brazil). A total of 74 stool samples were
evaluated in each group by the duplex PCR assay. This study was ap-
proved by the CEP Fiocruz/IOC under the number 1.308.312.
Pregnant women with or without HIV infection were chosen be-
cause of their depressed immune system, which would favor the sur-
vival and multiplication of Y. enterocolitica.
The DNA from stool samples was extracted using a DNA extraction
kit (QIAamp® DNA Stool Mini Kit; Qiagen), following the manufac-
turer's guidelines.
Regarding the identification of stool isolates in the culture media,
1 g of stool sample was added to 9 ml of cold mannitol broth (Peixotto
et al., 1979) and the enrichment was performed at 4 °C for 21 days.
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Journal of Microbiological Methods 154 (2018) 107–111
Then, the obtained growth suspension was recovered by direct plating
on CIN (Oxoid, Basingstoke, Hants, UK) and MacConkey (Oxoid)
medium and incubated at 37 °C for 18–24 h. Identification, biotyping,
and serotyping were performed according to Rusak et al. (2014).
Regarding the statistical analysis, Fisher's exact test was used for
analyzing proportions to evaluate the detection efficiency of the two
diagnostic methods, i.e., the duplex PCR assay and the culture-based
method (Boyapalle et al., 2001). All statistical analyses were performed
using the Rcmdr: R commander. R package version 2.5–1. (Fox and
Bouchet-Valat, 2018).
3. Results and discussion
All the 88 Yersinia strains analyzed in this study showed the pre-
sence of the tufA gene; thus, this gene could be a useful target in di-
agnostic purposes for the identification of Yersinia species (Table 1).
The amplification of a 405-bp rfbC fragment only in the Y. en-
terocolitica serotype O:3 samples indicated that this gene is an excellent
serotype O:3 marker for diagnostic purposes (Table 1). The duplex PCR
assay was able to detect other Y. enterocolitica serotypes than the O:3,
comprising all serotypes that cause diseases in humans and animals
(Drummond et al., 2012). The assay could also detect other Yersinia
species such as Y. kristensenii, Y. frederiksenii, Y. ruckeri, and the pa-
thogenic Y. pseudotuberculosis. In addition, the negative control strains
did not exhibit cross-reaction with the duplex PCR assay, a result that
reinforces the usage of this assay for the detection or identification of
Yersinia that causes gastroenteritis.
The BLASTN search in the NCBI database for the rfbC sequenced
target fragments revealed full-length identity only with the Y. en-
terocolitica serotype O:3 sequences. This finding is in accordance with
the duplex PCR results, thus confirming the specificity of this gene for
the identification of the serotype O:3. The rfbC gene located within the
rfb cluster is involved in colonization and invasion processes because it
is responsible for the biosynthesis of the O side chain in the LPS of
pathogenic strains of Y. enterocolitica (Asadishad et al., 2013).
Multiple alignments of the tufA primers with the closest tufA se-
quenced fragments within the Yersinia genus are depicted in Fig. 2. The
Y. similis strain 288 genome did not present a region that aligns with the
forward primer (Fig. 2A). The part of the genome of the strain 288 that
aligned with the reverse primer presented a huge divergence in the
nucleotide sequence (Fig. 2B). The lack of the region that matches with
the forward primer and the high diversity of the region that matches
with the reverse primer could be justified by the evolutionary distance
between Y. enterocolitica and Y. similis within the Yersinia genus. Whole-
genome sequencing studies placed these two species in antagonistic
branches in the phylogenetic tree of the Yersinia genus (Reuter et al.,
2014).
The tufA gene sequences of Y. enterocolitica strain CCUG 4586, Y.
frederiksenii strain CCUG 8246, and Y. mollaretii ATCC 43969 demon-
strated only one nucleotide diversity from the forward primer (Fig. 2A).
Single nucleotide polymorphism was expected for the tufA gene, which
has nucleic acid intragenomic identity levels ranging from 83.8% to
91.7% for Yersinia strains (Isabel et al., 2008). On the other hand, with
the exception of Y. similis strain 288, all other sequences analyzed
presented 100% nucleic acid similarity with the reverse primer
(Fig. 2B).
The genomes of Y. pestis strain Nairobi and Y. pseudotuberculosis
strain PA3606 presented 100% amino acid similarity with two primers
(Fig. 2A and B). The alignment result and the result of the duplex PCR
assay that amplified the tufA fragment of Y. pseudotuberculosis IAL1791
(Table 1) suggested that this assay could detect all three human pa-
thogenic Yersinia species (Drummond et al., 2012).
Nine sequences demonstrated 100% nucleic acid similarity with two
primers (Fig. 2A and B). All the results suggested that the duplex PCR
assay is able to detect Y. enterocolitica serotype O:3, various serotypes of
Y. enterocolitica, Y. kristensenii, Y. frederiksenii, Y. rohdei, Y.
L.A. Rusak et al. Journal of Microbiological Methods 154 (2018) 107–111

Fig. 2. Multiple alignment of the tufA primers and the closest tufA sequenced fragments within the Yersinia genus. A) Alignment with forward tufA primer. B)
Alignment with reverse tufA primer. A = adenine; C = cytosine; G = guanine; T = thymine; R = G or A.

entomophaga, Y. mollaretii, Y. intermedia, Y. ruckerii, Y. pseudotubercu-
losis, and Y. pestis.
Multiple alignments of the tufA primers with the closest tufA se-
quenced fragments out of the Yersinia genus are shown in Fig. 3. The
forward tufA primer presented 100% nucleic acid similarity with Ser-
ratia proteamaculans, S. liquefaciens, S. plymuthica, Serratia spp., Hafnia
alvei, Obesumbacterium proteus, and Enterobacter spp. (Fig. 3A). Al-
though the tufA primers have been designed in well-conserved regions
within the Yersinia genus, the two copies of the tuf gene (tufA and tufB),
found in enterobacteria, share high levels of identity and revealed
highly similar nucleic acid sequences that differ by < 1.4% (Dalmasso
et al., 2014; Isabel et al., 2008; Paradis et al., 2005). However, the tuf
gene still represents a good target for distinguishing phenotypically
close enterobacteria belonging to different genetic species (Paradis
et al., 2005).
In terms of tufA reverse primer, only H. alvei and O. proteus pre-
sented 100% nucleic acid similarity (Fig. 3B). Isabel et al. (2008)
conducted a phylogenetic study that constructed phylogenetic trees
based on tufA and tufB nucleic acid sequences from Yersinia and non-
Yersinia enterobacterial strains, which revealed that the members of the
H. alvei and O. proteus clade exhibited an intermediate intragenomic tuf
sequence level of divergence, in comparison with Yersinia genus and
other non-Yersinia enterobacterial species. The 16S rRNA gene se-
quence analysis showed that H. alvei is a member of the En-
terobacteriaceae family most closely related to Yersinia genus, which
could explain the alignment result (Ibrahim et al., 1993).
The results of this study demonstrated that the duplex PCR assay
could be a useful tool for the detection of Y. enterocolitica serotype O:3
when it presents the two amplified PCR fragments, the genus (tufA),
and the serotype O:3 (rfbC) markers. In the case of only the tufA frag-
ment amplification, it suggests that this sample or the strain could be Y.
enterocolitica of another serotype or even other Yersinia species.
However, in this case, we recommend another identification step, in an
attempt to avoid a possible cross-reaction with H. alvei and O. proteus.
Several detection and identification methods that use sequencing
have been developed and demonstrated good results; however, these
assays are more time-consuming and expensive than a single PCR assay
(Dalmasso et al., 2014; Neubauer et al., 2000).
The identification and detection systems based on the tufA and rfbC
genes exhibit various advantages over other systems that target

Fig. 3. Multiple alignment of the tufA primers and the closest tufA sequenced fragments out of the Yersinia genus. A) Alignment with forward tufA primer. B)
Alignment with reverse tufA primer. A = adenine; C = cytosine; G = guanine; T = thymine; R = G or A.

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L.A. Rusak et al.
chromosomal and plasmidial virulence markers such as ail, inv, yst,
yadA, and virF (Jourdan Alissa and Johnson, 2000; Thoerner et al.,
2003; Ye et al., 2014). Those markers (tufA and rfbC) are conserved in
nonpathogenic and pathogenic Yersinia strains, detecting strains lacking
these virulence genes (ail, inv, yst, yadA, and virF).
Regarding the results of the duplex PCR assay using the DNA from
stool samples and the identification of stool isolates in culture, the rfbC
fragment was not amplified either in the HIV-positive or in the -nega-
tive pregnant women group, which suggests the absence of Y. en-
terocolitica serotype O:3. Moreover, the tufA fragment was amplified in
14 (18%) stool samples in each group, which suggests the presence of
Yersinia spp. This duplex PCR assay demonstrated better detection ac-
curacy than the traditional culture-based methods; through culture of
samples from the HIV-negative pregnant women group (tufA positives),
only one isolate of Y. frederiksenii and one isolate of Y. enterocolitica
serotype O:7 were obtained. No Yersinia spp. was found through culture
of samples from the HIV-positive pregnant women group (even the tufA
positives). This result can be justified by the fact that Y. enterocolitica
has the ability to persist in a sample in a viable, but nonculturable, state
(Alexandrino et al., 2004). There was a significant difference in the
detection of Yersinia spp. (p < 0.001) between these two methods (the
duplex PCR assay and the culture-based method). The duplex PCR assay
was more efficient in detecting Yersinia spp. in stool samples than the
culture-based method (OR = 16.9; IC 95% [4.124–149.32]) in an order
of 16 times, which reinforces the premise of better detection accuracy
of the duplex PCR assay.
The duplex PCR assay proved to be a suitable method for the rapid
detection of Y. enterocolitica serogroup O:3, which has currently become
the dominant Yersinia species worldwide (Batzilla et al., 2011). This
assay is easy to perform, much faster than detection by cultivation, and
could be performed in any laboratory having a PCR apparatus.
In conclusion, we developed the duplex PCR assay that can be used
as a rapid screening method for the direct detection of Y. enterocolitica
serotype O:3, other serotypes, and other Yersinia species using total
extracted DNA from a source. This assay could also be used for the
precise identification of Y. enterocolitica serotype O:3. However, further
identification steps are needed for the identification of other Yersinia
genus members. We anticipate that this technique could be a useful tool
for hospital and veterinary surveillance studies on Yersinia worldwide.
Acknowledgments
We thank Professor Juliana Pfrimer Falcão from the collection of the
Brazilian Reference Center on Yersinia spp. other than Y. pestis (School
of Pharmaceutical Sciences of Ribeirão Preto, USP, Ribeirão Preto, São
Paulo State, Brazil) for kindly providing 15 Y. enterocolitica serotype
O:3 DNA samples for this study. This work was supported by Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
(FAPERJ) and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq).
Declaration of interest
None.
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