Int. J. Mol. Sci. 2015, 16, 25141-25153; doi:10.

3390/ijms161025141
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article

Detection and Identification of Probiotic

Lactobacillus plantarum Strains by Multiplex PCR Using
RAPD-Derived Primers
Alex Galanis 1,*, Yiannis Kourkoutas 1, Chrysoula C. Tassou 2 and Nikos Chorianopoulos 2,*

1
Department of Molecular Biology and Genetics, Democritus University of Thrace,
Alexandroupolis 68100, Greece; E-Mail: ikourkou@mbg.duth.gr
2
Institute of Technology of Agricultural Products, Hellenic Agricultural Organization-DEMETER,
Sof. Venizelou 1, Lycovrissi, Attiki 14123, Greece; E-Mail: ctassou@nagref.gr

* Authors to whom correspondence should be addressed;

E-Mails: agalanis@mbg.duth.gr (A.G.); nchorian@nagref.gr (N.C.);
Tel.: +30-25-5103-0634 (A.G.); +30-21-0284-5940 (N.C.);
Fax: +30-25-5103-0624 (A.G.); +30-21-0284-0740 (N.C.).

Academic Editor: Alejandro Cifuentes

Received: 9 September 2015 / Accepted: 16 October 2015 / Published: 22 October 2015

Abstract: Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are
two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both
display significant potential for the production of novel probiotic food products. The aim
of the present study was the development of an accurate and efficient method for the
molecular detection and identification of the above strains in a single reaction. A multiplex
PCR assay was designed for each strain, based on specific primers derived from Random
Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region
(SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB
strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay
was also successfully applied for the detection of the above cultures in yogurt samples
prepared in our lab. The proposed methodology may be applied for monitoring the
presence of these strains in food products, thus evaluating their probiotic character.
Moreover, our strategy may be adapted for other novel LAB strains with probiotic
potential, thus providing a powerful tool for molecular discrimination that could be
invaluable to the food industry.
Int. J. Mol. Sci. 2015, 16 25142

Keywords: probiotics; lactic acid bacteria; Lactobacillus plantarum; multiplex PCR;

RAPD; SCAR

1. Introduction

Probiotics are living microorganisms, with bifidobacteria and lactic acid bacteria (LAB)
constituting the most common microbial groups. According to the Food and Agriculture Organization
(FAO) and the World Health Organization (WHO) of the United Nations, probiotics are defined as
“live microorganisms which when administered in adequate amounts confer a health benefit on the
host” [1]. The theory that regular consumption of lactic acid bacteria in fermented foods may
contribute to enhancing health and longevity was originally developed by the Russian immunologist
and Nobel Laureate in Medicine Elie Metchnikoff and was presented in his book “The prolongation of
life”, published in 1907. Nowadays, probiotics represent a key fast growing section of the food
industry and are added to a number of products on the market, including yogurts, cheeses, ice creams
and other desserts [2]. According to a market report, probiotics’ demand worldwide was worth $27.9
billion in 2011 and is estimated to reach $44.9 billion in 2018, increasing at an annual growth rate of
6.8% from 2013 to 2018 [3]. Their strong market development and immense future potential are
mainly due to numerous studies that have linked consumption of probiotic beverages, foods, and
supplements to beneficial effects for consumers’ health [4]. Indeed, a probiotic-rich diet has been
connected with the prevention and potential treatment of several digestive disorders, such as irritable
bowel syndrome [5], necrotizing enterocolitis (NEC) in neonates [6], and ulcerative colitis [7]. Recent
studies employing in vitro and in vivo systems have also indicated that probiotic treatment might
reduce the risk for colon, liver, and bladder cancers [8]. It may also be effective against diet-induced
obesity through the modulation of genes associated with metabolism and inflammation in the liver
and adipose tissue [9], and even be therapeutically beneficial for the treatment of anxiety and
depression [10].
It is evident that isolation and characterization of new LAB strains with probiotic potential attracts
significant interest by the food industry for the development of novel functional food products. In this
context, two Lactobacillus plantarum strains, termed as 2035 [11] and ACA-DC 2640 [12] were
isolated from Feta cheese. Both exhibit probiotic potential and biotechnologically important
characteristics, as demonstrated by several in vitro tests, such as bile salt tolerance, ability to grow at
low pH, antimicrobial activity, antibiotic resistance etc. [12–15]. However, to further evaluate the
properties of these strains and establish their probiotic character, verification of their presence and
monitoring of their levels in food products are required. For this reason, an accurate method of strain
detection and identification is needed.
Conventional microbiological tests for phenotypical characterization are considered inadequate,
as they have limitations in discriminating large numbers of isolates with similar physiological
characteristics. Several DNA-based techniques have been developed to overcome this obstacle [16].
For example, the Pulsed Field Gel Electrophoresis (PFGE) methodology is commonly used for
probiotic strain differentiation and discrimination [17,18]. However, it cannot be applied for direct
Int. J. Mol. Sci. 2015, 16 25143

detection of a particular strain, in a single reaction [17]. Moreover, it is laborious, time-consuming and,
thus, inappropriate for large scale screening experiments in vivo, especially when microbial groups,
other than those needed to be identified, are at higher population levels. On the other hand, the
multiplex PCR methodology is commonly employed for efficient single step detection of LAB strains
in probiotic products [19–21]. It is easy to implement, fast, cost efficient, and requires a small amount
of template DNA. Simultaneous amplification of two or more loci in the same reaction with strain
specific primers that target the variable regions of the 16S rRNA, the hsp60 or other universal genes is
performed, thus increasing the sensitivity and accuracy of discrimination. However, when sequence
information of the target bacteria is not available, the design of specific primers by comparative
sequence analysis is not feasible. To overcome this limitation, the random amplified polymorphic
DNA (RAPD) technique may be applied. RAPD is a PCR-based assay that uses short arbitrary primers
that anneal to multiple random target sequences to generate the needed polymorphism [22,23].
The revealed polymorphic sites, namely sequence characterized amplified regions (SCAR), are then
used for the development of longer, sequence complementary primers. A more stringent annealing
temperature than that used with the RAPD analysis is also applied for the novel primers, thus
increasing the specificity and reproducibility of the assay.
The aim of the present study was the development of an assay for efficient and accurate molecular
detection of L. plantarum strains 2035 and ACA-DC 2640 in a single reaction. RAPD SCAR analysis
was firstly applied for the development of strain-specific primers as no sequence data was available for
either of the two strains.

2. Results and Discussion

2.1. Screening of RAPD Primers and Isolation of SCAR Markers

A total of 120 arbitrary primers were tested with RAPD PCR with DNA extracted from pure
cultures of L. plantarum strains 2035 and ACA-DC 2640. Thirty-one primers for L. plantarum 2035
and 37 for L. plantarum ACA-DC 2640 produced more than four scorable bands and were chosen
for further analysis. Out of the primers selected, primers RAPD-30 and RAPD-29 gave unique
RAPD profiles for L. plantarum 2035 and L. plantarum ACA-DC 2640, respectively (Figure 1).
The experimental procedure was repeated at least three times with the same conditions to confirm the
reproducibility of the RAPD analysis. A PCR product of 179 bp for L. plantarum 2035 (Figure 1Α)
was isolated from the agarose gel, cloned into an appropriate pBlueScript vector, and sequenced.
Similarly, in the case of L. plantarum ACA-DC 2640, a 401 bp band was considered as potential
strain-specific RAPD marker and isolated for further analysis (Figure 1Β).
Int. J. Mol. Sci. 2015, 16 25144

Figure 1. RAPD analysis for L. plantarum strains 2035 and ACA-DC 2640.
(Α) Electrophoretic profile generated with RAPD primer RAPD-30, of L. plantarum 2035
(lane 1), and 6 L. plantarum wild type strains (lanes 2–7). Lanes: 2, L. plantarum 141;
3, L. plantarum E95; 4, L. plantarum E106B; 5, L. plantarum E128; 6, L. plantarum E89;
7, L. plantarum E119. M: 1 kb DNA ladder. The numbers on the left of the figure indicate
the DNA size markers in base pairs (bp). The PCR product of 179 bp that was isolated
from the agarose gel for further analysis is indicated with the arrow; (Β) Electrophoretic
profile generated with RAPD primer RAPD-29, of L. plantarum ACA-DC 2640
(lane 1), and 6 L. plantarum wild type strains (lanes 2–7). Lanes: 2, L. plantarum 141; 3,
L. plantarum E95; 4, L. plantarum E106B; 5, L. plantarum E128; 6, L. plantarum E89; 7,
L. plantarum E119. M: 1 kb DNA ladder. The numbers on the left of the figure indicate the
DNA size markers in base pairs (bp). The PCR product of 401 bp that was isolated from
the agarose gel for further analysis is indicated with the arrow.

2.2. RAPD-Derived Primers for L. plantarum 2035 and L. plantarum ACA-DC 2640

The nucleotide sequences of the RAPD markers were used to design novel and potentially
strain-specific primers for L. plantarum strains 2035 and ACA-DC 2640 (Figure 2). The nucleotide
sequences for the primers of L. plantarum 2035 were: Forward primer (p30F): 5′-GTGATCGCAGTT
GGAAAACTG-3′, and Reverse primer (p30R): 5′-GTGATCGCAGGGAGATTATC-3′. The primers
were designed to the 5′ and 3′ ends of the obtained sequences (Figure 2Α). The nucleotide sequences
for the primers of L. plantarum ACA-DC 2640 were: Forward primer A (p29AF): 5′-GGGTAACGCC
ACAAGAAGC-3′, Forward primer B (p29BF): 5′-GTACTTGGCTAGTCGGCAG-3′ and Reverse
primer (p29R): 5′-GGGTAACGCCTTGCACTTTG-3′. The primers p29AF and p29R target the 5′ and
3′ ends of the RAPD marker, respectively, whereas the primer p29BF targets an internal potentially
polymorphic region (Figure 2Β). A set of primers (Lac1F and Lac2R) that recognizes the 16S rRNA
gene of all Lactobacillus strains and generates a 340 bp product was also comprised as a positive
control marker [24]. The specificity of the primers and the estimated size of PCR products are
presented in Table 1. Optimization of the reactions was performed by adjusting the amount of all
Int. J. Mol. Sci. 2015, 16 25145

primers, the concentration of dNTPs and MgCl2, and the cycling conditions (extension and annealing
time and temperature and number of cycles) (data not shown) [25].

(A)

(B)

Figure 2. Nucleotide sequences of RAPD amplicons. (A) Nucleotide sequences of RAPD

amplicons specific to L. plantarum 2035. Arrows represent forward primer p30F and
reverse primer p30R and the underlined sequence belongs to the oligo decamer primer
RAPD-30; (Β) Nucleotide sequences of RAPD amplicons specific to L. plantarum
ACA-DC 2640. Arrows represent forward primer p29AF and reverse primer p29R and the
underlined sequence belongs to the oligodecamer primer RAPD-29. The sequence of the
forward primer p29BF is in bold italics.

Table 1. Specificity of primer pairs for Multiplex PCR on L. plantarum 2035 and
L. plantarum ACA-DC 2640. The expected size of PCR products is indicated.
Specificity of Primer Pairs
Reference Strain
p30F/p30R (179 bp) p29AF/p29R (401 bp) p29BF/p29R (200 bp) Lac1F/Lac1R (340 bp)
L. plantarum 2035 + − − +
L. plantarum ACA-DC 2640 − + + +

2.3. Multiplex PCR with RAPD-Derived Strain Specific Primers

The multiplex PCR was initially tested with DNA extracted from L. plantarum 2035 and five other
L. plantarum strains. As presented in Figure 3Α, L. plantarum 2035 gave two distinct products (179
and 340 bp), whereas only one, the 340 bp positive control product, was produced for the five other
L. plantarum strains tested (Figure 3A). In a similar way, a multiplex PCR was performed with DNA
from L. plantarum ACA-DC 2640 and four other L. plantarum strains. Significantly, only three PCR
products were produced (401, 340 and 203 bp) for L. plantarum ACA-DC 2640 promoting the
specificity of the reaction (Figure 3Β).
Int. J. Mol. Sci. 2015, 16 25146

Figure 3. Agarose gel electrophoresis of PCR products from multiplex PCR assay.
(A) Lanes: 1, L. plantarum 2035; 2, L. plantarum 141; 3, L. plantarum E95; 4,
L. plantarum E106B; 5, L. plantarum E128; 6, L. plantarum E89. M: 1 kb DNA ladder. A
PCR product of 179 bp was generated from the primer set p30F/p30R, whereas a PCR
product of 340 bp was generated from the primer set Lac1F/Lac1R. Both products are
indicated with the corresponding arrows; (B) Lanes: 1, L. plantarum ACA-DC 2640;
2, L. plantarum 141; 3, L. plantarum E95; 4, L. plantarum E106B; 5, L. plantarum E128.
M: 1 kb DNA ladder. Two PCR products of 401 bp and 200 bp were generated from
the primer sets p29AF/p29R and p29BF/p29R, respectively, and are indicated with
the corresponding arrows. A PCR product of 340 bp was generated from the primer
set Lac1F/Lac1R.

To further validate the specificity of the RAPD-derived primers for L. plantarum strains 2035 and
ACA-DC 2640, the multiplex PCR assay was also tested with DNA extracted from 17 L. plantarum as
well as from six other LAB strains, including the reference strains L. casei Shirota ACA-DC 6002,
L. rhamnosus GG ATCC 53103 and L. casei ATCC 393, which are commonly used in probiotic
products. Consistently, the primer pair p30F and p30R generated a distinct product only for
L. plantarum 2035 (Table 2). Similarly, the specificity of the novel primers for L. plantarum ACA-DC
2640 was positively tested in a total of 23 LAB strains as indicated in Table 2.

Table 2. The complete list of Lactobacillus strains tested in multiplex PCR with the
RAPD-derived primer pair p30R/p30F for L. plantarum 2035 and primer pairs
p29AR/p29F and p29BR/p29F for L. plantarum ACA-DC 2640, respectively. A unique
2-band pattern is only produced for L. plantarum 2035 and a unique 3-band pattern is only
produced for L. plantarum ACA-DC 2640 (indicated as +).
Strain Tested 2-Band Pattern 3-Band Pattern
Lactobacillus plantarum 2035 + −
L. plantarum ACA-DC 2640 − +
L. plantarum E1 − −
L. plantarum E4 − −
L. plantarum E45 − −
L. plantarum E50 − −
L. plantarum E66 − −
L. plantarum E68 − −
L. plantarum E71 − −
Int. J. Mol. Sci. 2015, 16 25147

Table 2. Cont.
Strain Tested 2-Band Pattern 3-Band Pattern
L. plantarum E73 − −
L. plantarum E77 − −
L. plantarum E79 − −
L. plantarum E10 − −
L. plantarum E69 − −
L. plantarum E282 − −
L. plantarum E63 − −
L. plantarum E146 − −
L. plantarum E287 − −
L. pentosus Ε110 − −
L. pentosus 281 − −
L. casei Shirota ACA-DC 6002 − −
L. casei ATCC 393 − −
L. rhamnosus GG ATCC 53103 − −
L. delbrueckii subsp. bulgaricus − −

Figure 4. Molecular identification of L. plantarum 2035 or L. plantarum ACA-DC 2640 in

food products, using the multiplex PCR assay. Lanes 1–4 correspond to yogurt samples
prepared in the lab containing either L. plantarum 2035 (lanes 1 and 3) or L. plantarum
ACA-DC 2640 (lanes 2 and 4); Lane 5 that corresponds to a sample prepared in the lab
lacking L. plantarum 2035 or L. plantarum ACA-DC 2640 served as a negative control.
The experiment was repeated three times with different yogurt preparations.

Finally, to test the applicability of our method in food products, yogurts were prepared in the lab
using a commercial yogurt culture containing either L. plantarum 2035 (samples 1 and 3) or
L. plantarum ACA-DC 2640 (samples 2 and 4). All samples were mixed with sterilized ¼ Ringer
solution, total DNA was extracted, and multiplex PCR was implemented using the novel strain-specific
primers. Two PCR products corresponding to the unique pattern of L. plantarum 2035 (Figure 3A)
Int. J. Mol. Sci. 2015, 16 25148

were generated for samples 1 and 3 (Figure 4). Accordingly, three PCR products, corresponding to the
unique pattern of L. plantarum ACA-DC 2640 (Figure 3B), were generated for samples 2 and 4
(Figure 4), thus confirming the presence of the specific strains in the above samples. We are currently
preparing novel dairy (cheese) and non-dairy (sausages) food products containing either L. plantarum
2035 or L. plantarum ACA-DC 2640 and are investigating their properties both in vitro and in vivo.

3. Experimental Section

3.1. Bacterial Strains and Culture Conditions

L. plantarum 2035 was kindly provided by K. Koutsoumanis, Laboratory of Food Microbiology and
Hygiene, Dept. of Food Science & Technology, Aristotle University, Thessaloniki, Greece.
L. plantarum ACA-DC 2640 and L. casei Shirota ACA-DC 6002 strains were kindly provided by
E. Tsakalidou, Laboratory of Dairy Research, Agricultural University of Athens. L. rhamnosus GG
ATCC 53103 and L. casei ATCC 393 strains were obtained from DSMZ (Braunschweig, Germany).
All other Lactobacillus strains tested were obtained from the collection of the Institute of Technology
of Agricultural Products, Hellenic Agricultural Organization-DEMETER, Athens, Greece. All LAB
strains were grown anaerobically at 37 °C on MRS broth (Merck, Darmstadt, Germany).

3.2. DNA Extraction from Pure Cultures

Extraction of genomic DNA from a pure culture was performed using a DNeasy Tissue Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s protocol. To determine the amount of extracted DNA,
the absorbance at 260 nm was measured using a UV spectrophotometer (Eppendorf, Hamburg, Germany).

3.3. Yogurt Production

Yogurts were manufactured by inoculating pasteurized bovine milk (100 mL) with a commercial
yogurt culture CH-1 (Chr. Hansen, Hørsholm, Denmark) consisting of Strep. salivarius ssp.
thermophilus and L. delbrueckii subsp. bulgaricus along with L. plantarum 2035 or ACA-DC 2640.
Τhe initial cell counts for L. plantarum 2035 or ACA-DC 2640 were approximately 8–9 log cfu·g−1.

3.4. Microbial Enumeration

Lactobacilli (gram-positive, catalase-negative) counts were measured after serial diluting

and plating on acidified MRS agar (Fluka, Buchs, Switzerland) at 37 °C for 48 h anaerobically
(Anaerobic Jar, Anerocult C, Merck, Germany).

3.5. DNA Extraction from Yogurt Samples

Ten grams of a yogurt sample were blended with 90 mL of sterilized ¼ Ringers solution
(Sigma-Aldrich, Gillingham, UK). Total DNA extraction from the liquid suspension was performed
using a DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
Int. J. Mol. Sci. 2015, 16 25149

3.6. RAPD PCR

All decamer RAPD primers used in this study were designed using the RAPD primer generator
application (Available online: http://www2.uni-jena.de/biologie/mikrobio/tipps/rapd.html) (J. Wöstemeyer,
Institute of General Microbiology and Microbial Genetics, Jena, Germany) and obtained from
VBC-Biotech, Wien, Austria. PCR reactions were performed in a total volume of 20 μL, containing
2 units Taq DNA polymerase (Kapa Biosystems, Wilmington, NC, USA), 2 μL of 10× PCR buffer
(without MgCl2), 200 μM each dNTPs (Kapa Biosystems), 2.5 mM MgCl2 (Kapa Biosystems), 100 ng
template DNA, and 10 pmol of selected primer. Amplifications were performed in a Thermal Cycler
(Mastercycler Eppendorf, Hamburg, Germany) under the conditions: initial denaturation at 95 °C for
5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, primer annealing for 1 min at 36 °C,
and primer extension for 1.5 min at 72 °C with a final extension period at 72 °C for 5 min. Then,
samples were cooled to 4 °C. Aliquots (5.0 μL) of the amplified products were subjected to
electrophoresis in 1.5% w/v agarose gels in TAE buffer. The gels were stained with 0.5 μg·mL−1
ethidium bromide, visualized under UV illumination, and photographed with a digital camera (Gel Doc
EQ System, BioRad, Milan, Italy). The PCR reactions were repeated three times for each RAPD
primer, and those that demonstrated reproducibility were considered for further marker isolation
and cloning.

3.7. Cloning and Sequencing

After electrophoresis separation of the RAPD fragments, the chosen bands were excised from the
gel. DNA isolation was performed using a NucleoSpin Extract II kit (MACHEREY-NAGEL, Düren,
Germany) according to the manufacturer’s instructions. The potential strain-specific RAPD markers
were cloned into the pBlueScriptSK + vector following the TA cloning protocol described by Zhou
and Gomez-Sanchez [26]. Briefly, the purified DNA fragments were incubated with 2 units of Taq
polymerase (Kapa Biosystems), at 72 °C for 30 min in the presence of the four dNTPs, 10 mM each
dNTP (Kapa Biosystems), for the addition of 3′-A overhangs, followed by purification using
a NucleoSpin Extract II kit (MACHEREY-NAGEL, Düren, Germany). Accordingly, the plasmid was
firstly digested with the restriction enzyme EcoRV (Fermentas, MA, USA) to generate unique
blunt-ends in the multiple cloning sites of the polylinker. Then, the blunt-ended plasmid DNA was
purified using a NucleoSpin Extract II kit (MACHEREY-NAGEL) and incubated with 5 units of Taq
polymerase (Kapa Biosystems) at 72 °C for 2 h, in the presence of 100 mM TTPs (Kapa Biosystems)
for the addition of 3′-T overhangs. The T-tailed plasmid DNA was then purified using a NucleoSpin
Extract II kit (MACHEREY-NAGEL) and incubated overnight at 16 °C with 3 units of T4 DNA ligase
(Fermentas) to distinguish from the vectors without T-tails. T-vectors were separated from the
self-ligated and concatemerized plasmid DNA on a 1% w/v agarose gel. The extraction of the T-vector
band was performed using a NucleoSpin Extract II kit (MACHEREY-NAGEL). The A-tailed DNA
fragment was ligated into the T-vector and then transformed into Escherichia coli Top-10 competent
cells by heat shock method according to Sambrook et al. [27]. The cells were then spread on
Luria-Bertani (LB) medium containing appropriate ampicillin, IPTG, and X-gal and incubated at 37 °C
for 16 h. Clone selection was carried out based on blue/white selection. Extraction of the recombinant
Int. J. Mol. Sci. 2015, 16 25150

plasmids was performed using a NucleoSpin Plasmid kit (MACHEREY-NAGEL) and checked for
insert by diagnostic digest with HindIII (Fermentas) and EcoRI (Fermentas). The resulting clones were
sent for sequencing to VBC-Biotech, Austria. The nucleotide sequence of the cloned fragment was
determined from three clones. The nucleotide sequence of the cloned fragment was determined from
three clones. To design the forward and reverse primers, the decamer primer was extended by a few
nucleotides (8–10 bases) based on the obtained sequence. The Tm, GC, and other factors were
analyzed using Primer3, primer design program (Available online: http://bioinfo.ut.ee/primer3/) and
the final compatible forward and reverse primers selected for specific SCAR amplification. NCBI
Primer-BLAST (Available online: http://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to evaluate
the specificity of the designed primers.

3.8. Multiplex PCR

Multiplex PCR reactions were performed in a total volume of 20 μL, containing 2 units of Taq
DNA polymerase (Kapa Biosystems), 2 μL of 10× PCR buffer (without MgCl2), 200 μM each dNTPs
(Kapa Biosystems), 2.5 mM MgCl2 (Kapa Biosystems), and 100 ng template DNA. The specific
designed primers of L. plantarum 2035 consisted of p30F: 5′-GTGATCGCAGTTGGAAAACTG-3′
(10 pmol), and p30R: 5′-GTGATCGCAGGGAGATTATC-3′ (10 pmol) and of L. plantarum ACA-DC
2640 consisted of p29AF: 5′-GGGTAACGCCACAAGAAGC-3′ (8 pmol), p29BF: 5′-GTACTTGG
CTAGTCGGCAG-3′ (8 pmol) and p29R: 5′-GGGTAACGCCTTGCACTTTG-3′ (16 pmol),
respectively. Additionally, a set of primers consisted of Lac1F: 5′-AGCAGTAGGGAATCTTCCA-3′
and Lac1R: 5′-ATTYCACCGCTACACATG-3′ that recognize region in the 16S rRNA gene of all
lactobacilli was also included in the reactions (8 pmol of each primer) as a positive control [24].
Amplification was performed in a Thermal Cycler (Mastercycler Eppendorf, Hamburg, Germany)
under the conditions: for L. plantarum 2035, 95 °C (2 min), followed by 24 cycles of 95 °C (30 s),
61 °C (30 s), 72 °C (60 s), followed by a final extension step at 72 °C (2 min); for L. plantarum
ACA-DC 2640, 95 °C (2 min), followed by 22 cycles of 95 °C (30 s), 63 °C (60 s), 72 °C (60 s),
followed by a final extension step at 72 °C (2 min). The PCR products were separated on 1% w/v
agarose gels, visualized under UV illumination, and photographed with a digital camera (Gel DocEQ
System, BioRad, Milan, Italy).

4. Conclusions

In this study, an efficient and accurate method for the detection of Lactobacillus plantarum strains,
termed as 2035 and ACA-DC 2640, in a single reaction, was presented. It is a novel multiplex PCR
assay utilizing strain-specific primers derived from RAPD analysis. The proposed methodology may
be used to assess the survival and the adhesion properties of the two strains in vivo and characterize
their probiotic character and applicability for the production of novel functional foods with potential
health benefits.
Int. J. Mol. Sci. 2015, 16 25151

Acknowledgments

This work has been co-financed by the European Regional Development Fund (ERDF) of the EU
and by National Resources under the Operational Program Competitiveness and Entrepreneurship
(EPAN II), Action “COOPERATION 2011”, Project “ProbioDairyMeat” (Project Nr. 11SYN_2_571)
and by the Hellenic Agricultural Organization-DEMETER, Project “Research and evaluation of
quality milk characteristics at responsibility areas of Patra”.

Author Contributions

Alex Galanis designed the studies, performed the experiments, and wrote the paper;
Nikos Chorianopoulos designed the studies and discussed the results; Yiannis Kourkoutas and
Chrysoula C. Tassou contributed the reagents, analyzed data and discussed the results.

Conflicts of Interest

The authors declare no conflict of interest.

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