Journal of the Venezuelan Society of

Microbiology

ISSN: 1317-973X
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Venezuela Venezuelan Society of Microbiology

Chacon, Luz; Barrantes, Kenya; García, Cristina; Achi, Rosario

Standardization of a PCR for detecting invA gene of Salmonella spp. lettuce Journal of the Venezuelan Society of
Microbiology, vol. 30, no. 1 January-June 2010, pp. 18-23
Venezuelan Society of Microbiology
Caracas Venezuela

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 Journal of the Venezuelan Society of Microbiology 2010; 30: 18-23
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Original article

Standardization of a PCR for detecting gene invA from Salmonella spp.

lettuce

Luz Chacon *, Kenya Barrantes, Cristina Garcia, Rosario Achí

INISA, University of Costa Rica.

Received 25 September 2009; accepted April 27, 2010

Summary: Salmonella spp. is a major bacterial pathogen causing diarrhea, which is transmitted both by the fecal-oral route, such as contaminated food and water. In
this work, a PCR was standardized in lettuce for gene detection invA from
Salmonella spp .; said gene is related to the process of invasion of the intestinal epithelium. With PCR developed in this work was achieved standardize a method that
allows gene amplification invA with detection of 10 2 CFU / 25 g. This method shortens the response times of the presumptive results and provides additional information
to traditional culture of the pathogen. The study of gene invA
It establishes the pathogenic potential of microorganisms present in the sample, which can be useful for public health.

Keywords: Salmonella spp., gene invA, PCR, lettuce

PRC Standardization of a method for the detection of the Salmonella spp. invA gene
in lettuce
Abstract: Salmonella spp. is a bacterial pathogen Very Important That causes diarrhea and Which is transmitted through the fecal-Both oral pathway, as by contaminated food
and water. In This study we standardized PCR method for the detection in lettuce of the Salmonella spp.
invA gene. This gene is related to the invasion of the intestinal epithelium process. With the PCR method developed In This study We were able to standardize a method
Which Permits the amplification of the invA gene with a 10 2 CFU / 25g detection. This method Shortens the response times of the presumptive results and complementary
information to the Gives traditional culture of the pathogen. The study of the
invA Establishes the gene of the potential pathogenic microorganism present in the sample, Which can be useful for public health purposes.

Keywords: Salmonella spp., invA gene, PCR, lettuce

* Correspondence:
E-mail: luz.chacon@ucr.ac.cr

Introduction Salmonella spp. It produces a variety of clinical pictures; the most

Salmonella spp., is the leading cause of bacterial foodborne enteritis
worldwide, according to data from the World Health Organization [1]. In
Austria 103 cases were filed per 100,000 inhabitants in 2003 [2]. In the
United States, it is estimated to occur about 1.4 million non-typhoid
infections, which generated 168,000 doctor visits, 15,000
hospitalizations and 580 deaths annually [3]. Data "World Health
Organization Global Salm-Surv Country data bank" between 2000 and
2002 for Costa Rica show 49 cases of salmonellosis associated with
human and 60 associated with animals, and Venezuela 691 cases of
salmonellosis associated with human and 162 associated animals [4].
common is gastroenteritis characterized by diarrhea and vomiting.
Transmission usually occurs by ingestion of contaminated food or
contact with infected animals. Typhoid, mainly caused by eating food
contaminated with Salmonella enterica serovar Typhi, is the most
severe form of enteric fever. It is characterized by prolonged fever,
bacteremia and spread to several organs. The table can have a
three-week course. Typhoid non bacteremia occurs mainly in young
children and the elderly and is characterized by intermittent post a
picture of gastroenteritis. In addition there is, after infection with this
bacterium that colonizes carrier state and gallbladder and maintained
by
 Chacon et al. / Journal of the Venezuelan Society of Microbiology 2010; 30: 18-23
long periods of time [5].
In developed countries, transmission Salmonella
It occurs mainly by consumption of animal products, particularly fresh
meat and eggs. By contrast, in developing the main sources of
pollution countries are fresh vegetables, water and person to person
transmission [6]. An important factor are the natural reservoir of the
bacteria; in so far as S. enterica serovar. Typhi and paratyphi are
unique serovars of human beings, other as Typhimurium and Litchfield,
can be considered as zoonotic, because they have between host
animals such as poultry and livestock [7-9]. An important gene in
virulence of Salmonella spp. is the invA. this gene is a virulence factor
related to the process of invasion of the intestinal epithelium and used
by other enteropathogens such as Shigella spp. during the infection
process. The Gen invA It is common in all invasive varieties, which
means that can be associated with possible vitriolic pictures. It is
encoded in the bacterial chromosome which gives greater stability in a
region known as pathogenicity island 1
Salmonella ( SPI1). This island encodes, among other proteins type III
secretion system [10-13]. The Gen invA It has been widely used in
studies to detect Salmonella spp. in food samples, mainly due to
stability having from genetically [14-16]. Detection Salmonella by
conventional culture it is considered the reference method. However,
this technique has disadvantages such as low sensitivity and time to
obtain a result. Currently they arise other diagnostic alternatives such
as molecular biology methods. One of the most used is the polymerase
chain reaction (PCR acronym), which allows detection of
microorganisms in food in less time compared with conventional culture
techniques. Among the limiting factors are the numerous PCR
inhibitory substances can be found in the food matrix (hemoglobin,
lactoferrin, polysaccharides and fats); Additional DNA detection of a
pathogen, does not ensure the viability of the sample when the
analysis, which represents a disadvantage with respect to conventional
cultivation. [17-19]. Moreover, the food culture isolation does not
ensure that the isolated microorganism possesses virulence
determinants necessary to cause infection and disease in a susceptible
host [20].
The main objective of this study was to obtain a PCR method
capable of directly identify genes associated with the virulence of Salmonella
spp., and in addition to traditional diagnosis of this pathogen tool, and
also provides important information in terms of public health in the
short term.
19
Materials and methods
Bacteria used: For processing lettuce samples and standardized PCR
was used a strain of Salmonella enterica serovar Enteritidis (ATCC
13076). For testing specificity of PCR the following bacteria were used: Esch
coli ATCC
25922, E. coli ( LT- / ST), E. coli ( LT + / ST), E. coli ( LT- / ST +),
E. coli ( LT + / ST +), E. coli enteropathogenic, Enterobacter cloacae,
Citrobacter freundii, phenylpyruvica Moraxella, Proteus mirabilis, Vibrio
cholerae, Pseudomonas aeruginosa, Brevundimonas tiny, plesiomonas
shigelloides, Aeromonas hydrophila, Staphylococcus aureus ATCC
22923, Shigella flexneri, S. flexneri ( PINV) and S. sonnei which belong
to the bacterial collection Health Research Institute at the University of
Costa Rica.
Sample description: In this study 13 samples of 25 g, in triplicate (39
total subsamples) butter lettuce variety analyzed (we used Lactuca
sativa var. capitata), supermarkets acquired in the Central Valley.
Sample processing: Portions of 25 g of lettuce were contaminated
intentionally with Salmonella
ATCC 13076 (serial dilutions of 10 7 CFU / mL to 10 1
CFU / mL). Additionally a portion of 25 g was used. with 1 mL of sterile
peptone water as negative control. These samples were subjected to a
pre-enrichment adding 225 mL of lactose broth and supplemented with
novobiocin lactose broth and incubated for 24 hours at 35 ° C. After
incubation was added 1 mL of this pre-enrichment in a conical sterile
Eppendorf tube for DNA extraction of the cell pellet, and one milliliter
he was placed in tetrathionate and selenite-cystine broth which was
incubated for 24 hours at 35 ° C. Finally a passage of the latter was
performed at media citrate deoxycholate (DC) and xylose lysine
desoxycholate (XLD), where suspicious colonies morphology were
chosen Salmonella spp., for analysis by conventional biochemical
methods.
Biochemical identification: tests for urea, mobility, indole, citrate and TSI
(triple sugar iron agar, for its acronym in English) were performed. the
API 20E BioMérieux® was then used to complete identification.
Isolates with profile Salmonella spp. They were serologically confirmed
with the antiserum Salmonella
O antiserum poly-AI & Vi (DIFCO).
DNA extraction: a fraction of 1 ml of pre-enrichment broth, both lettuce
contaminated with known concentrations of separated Salmonella spp.,
such as those mixed with sterile peptone water, which was centrifuged
for 10 minutes at 34,000 g. The supernatant was decanted, and the cell
pellet was resuspended in 1 mL of sterile distilled water. This washing
procedure with
twenty Chacon et al. / Journal of the Venezuelan Society of Microbiology 2010; 30: 18-23

distilled water was repeated 3 times to remove any PCR inhibitors.
DNA extraction was performed by thermal shock button resuspending
in 1 mL of distilled water and placing it in a container of water at 95 ° C
for 10 minutes. The tube was centrifuged for 10 minutes at 33,000 g,
and the supernatant DNA was separated in another sterile tube, which
was stored at -70 ° C. For the DNA used as positive control of PCR, it
was inoculated 1 ml of sterile distilled water with a colony of Salmonella
Abnormal hemoglobins and Allied Disorders (CIHATA) of the University
of Costa Rica.
PCR reproducibility: extracting DNA from 30 isolates was performed Salmon
spp. obtained Microbiology Lab Food and Water Microbiology Faculty
and the collection of microorganisms Health Research Institute (INISA),
University of Costa Rica. These are detailed in Table 2. For the
extraction technique thermal shock described above is applied.

spp. and the above method of centrifugation, washed and DNA

extraction was repeated heat.

Selection of the initiators: Salm3 initiators
5'GCTGCGGCGCAACGGCGAAG-3 'and 5'- Salm4
TCCCGGCAGAGTTCCCATT-3' [12, 15] were used. Its specificity
genome Salmonella enterica was corroborated by the BLAST (Basic
Local aligment Search Tool) program, showing for Salm3 an identity
equal to 20/20, (E-value = 0.006); and Salm4 identity = 19/19 (E-value
= 0.017).
PCR specificity: DNA extraction from bacteria indicated above using the
described technique was performed. The culture isolation was
performed on blood agar, tergitol 7 and agar DC incubating for 24
hours at 35 ° C. Enterobacteriaceae were identified with the API 20E
system and non-enteric strains ( S. aureus) They were identified as
reference protocol [22].

PCR: Struber it proposed by [12] and modified protocol was followed.
Among the amendments emphasizes the use of PCR touchdown; This
consisted of 10 cycles with a temperature of 65 ° C initial annealing,
decreasing 1 ° C each cycle; followed by 30 cycles with a temperature
stable hybridization at 55 ° C. Furthermore dimethylsulfoxide was
added as adjuvant. Tested protocols work and working conditions are
detailed in Table 1. The reactions were performed in a thermocycler
results
Standardizing the PCR: To determine the optimal concentration of
reagents for obtaining suitable amplification product of 389 bp of the
gene invA, It was required several PCR. It began with the
concentrations of reagents proposed by Struber et al [ 12]. Because the
amplification not performing achieved under these conditions, it was
necessary to change the temperatures and times in the thermocycler
and

Table 1. Tests on the standardization of the PCR technique for detecting gene invA in Salmonella spp.

Working protocols (ul)

reagents
1 2 3 4 5 6 7

10X buffer 1.0 1.0 1.0 1.0 1.0 1.0 1.0

MM MgCl2 / .mu.l 1.5 2.0 2.0 2.7 1.3 3.3 2.0

DMSO - - - - 0.15% 0.25% 0.25%

mM dNTPs / .mu.l 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Taq polymerase IU / .mu.l 0,025 0,025 0,025 0,025 0,025 0,025 0,025

Initiators ug / ul 4.0 x 10- 5 8.0 x 10- 5 2.0 x 10- 4 4.0 x 10- 4 4.0 x 10- 4 4.0 x 10- 4 1.0 x 10- 4

Gene Amp PCR System 2400, Perkin Elmer ®.
Visualization of amplicons: was performed by gel electrophoresis of 2%
agarose stained with ethidium bromide, the gel concentration was 0.5
mg / mL. The run was performed at 140 V for 45 minutes in TBE buffer
pH 8.3.
Purification and sequencing: The band of 389 bp obtained was purified
from agarose gel using a commercial kit (Qiagen Qiakit ®). This product
was sequenced using the same primers that touchdown PCR.
equipment ABI 310 (Applied Biosystems was used for sequencing ®) Research
Center
variations in concentration of MgCl 2. The different conditions assayed
for concentration of reactants are seen in Table 1. Variations with
respect to amplification protocol were directed to changes in the
annealing temperature of the primers in order to obtain a suitable
coupling to the target sequence, and optimal DNA polymerization. The
results were not optimal, so proceeded to use a variation of traditional
PCR, known as PCR touchdown. Reactant concentration and
temperature of the amplification is shown in Table 1, Procolo 7: Buffer
Tris-HCl pH 8.3 1X, MgCl 2 2.0 mM / uL, 0.2 mM / microL dNTPs, 1.0
x10- 3 ug / uL, 0.024 Taq IU / uL and 0.25% DMSO. In assessing the
 Chacon et al. / Journal of the Venezuelan Society of Microbiology 2010; 30: 18-23 twenty-one

Table 2. Strains used for standardization of the PCR technique for detecting gene invA in Salmonella
1 2 3 4 5 6 7 8 9 10 eleven
spp.

Salmonella 009 Salmonella 122

Salmonella 016 Salmonella 123

Salmonella 021 Salmonella 130 500 bp

400 bp
Salmonella 023 Salmonella 135

Salmonella 031 Salmonella 137

Salmonella 033 Salmonella 146

Salmonella 035 Salmonella 150

Salmonella enterica subsp. enterica

Salmonella 036
ATCC 6539, originally Typhi

Salmonella enterica subsp. enterica Figure 1. Gel electrophoresis with 2% agarose ethidium bromide staining for analysis
Salmonella 041
ATCC 14028, originally Paratyphi reproducibility Salmonella spp. Line 1-7:
Salmonella strains 16, 35, 53, 59, 83, 130 and 137, line 8: E. coli ATCC, line 9: Salmonella ATCC,
Salmonella enterica subsp. enterica
Salmonella 049 line 10: Control reagents, line 11: Molecular Weight Marker Fermentas ® SM0323.
ATCC 13076, originally Enteritidis

Salmonella 050 Escherichia coli ATCC 25922

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Salmonella 053 Shigella flexneri 397

Salmonella 059 Shigella flexneri ( OP) 500 bp

500 bp
Salmonella 073 Shigella sonnei 433
400bp
Salmonella 082 Enterobacter cloacae 296

Salmonella 083 Citrobacter freundii 294

Salmonella 087 Moraxella phenylpyruvica 136

Figure 2. Gel Electrophoresis 2% agarose stained with ethidium bromide for analysis
Salmonella 095 Proteus mirabilis 13 specificity Salmonella spp. Line 1 molecular weight marker; line 2 Plesiomonas shigelloides; line
3 Proteus mirabilis; line 4 Pseudomonas aeruginosa; Line 5 Positive control:
Salmonella 112 Pseudomonas aeruginosa 28

Salmonella 114
plesiomonas shigelloides
Brevundimonas tiny
Aeromonas hydrophila 35 Salmonella spp; line 6 Citrobacter freundii; line 7 Aeromonas hydrophila;
line 8 Brevundimonas diminuta; line 9 Moraxella phenylpyruvica; line 10 Staphylococcus
Escherichia coli 64111 aureus; line 11. Escherichia coli 64111; line 12. E. coli 286; line 13. E. coli 75688; line 14. E.
coli 34420; line 15. Control reagents; line 16. Marker Molecular Weight Fermentas ® SM0323.
Escherichia coli 286

Escherichia coli 75C88 Escherichia coli 34420

Staphylococcus aureus
PCR vs conventional culture: several portions of 25 g of lettuce
product in agarose gels 2%, a distinct band of 389 bp (Figure 1) was contaminated with known concentrations were analyzed by cultivation Salmo
observed. spp. and it was achieved by conventional breeding and show PCR
To ensure that the band observed actually correspond to gene invA from concentrations up to 10 2 CFU / 25 g. The extracted DNA lactose broth
Salmonella enterica the product obtained, which showed high identity supplemented with novobiocin showed no amplification product of 389
(281/282 or 99%, E-value = 2X10- sequenced 143) gene with invA in the bp of.
genomes of Salmonella enterica serovar Gallinarum,

Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Discussion

Typhi, Salmonella enterica serovar Typhimurium and Salmonella
enterica serovar Enteritidis, (database of the National Center for After several changes in base [12] protocol detection of a 389 bp
Biotechnology Information-NCBI), income numbers AM933173.1, product was obtained, the gene invA,
FM200053.1, AL627276.1, EU348365.1, and AM933172.1, specific Salmonella spp., using PCR variant
respectively). touchdown. This PCR technique was shown to be reproducible,
sensitive and specific for detection of Salmonella spp. It is
recommended that the standardization of a PCR is performed in each
Reproducibility of the PCR technique: DNA isolations analyzed 30 Salmonellalaboratory and for each matrix used; in our case, the original protocol
spp. (Identified biochemically and serologically) under standard PCR had to be modified considerably to be adapted to laboratory conditions.
protocol. In all cases the corresponding band was observed gene invA ( Figure
1).
The addition of DMSO allowed to separate secondary structures rich
in guanine-cytosine in the last cycles of a PCR reaction and is reported
Specificity of PCR: DNA of different bacteria was analyzed Salmonella spp., in the literature [21, 22]. Additionally, with the increased stringency of
no 389 bp band (Figure 2) observed. the reaction, produced by the increase
22 Chacon et al. / Journal of the Venezuelan Society of Microbiology 2010; 30: 18-23

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