ARTICLE IN PRESS

Systematic and Applied Microbiology 29 (2006) 49–58

www.elsevier.de/syapm

Monitoring Lactobacillus plantarum in grass silages with the aid of

16S rDNA-based quantitative real-time PCR assays
Michael Klockea,, Kerstin Mundta, Christine Idlera, Joseph McEniryb,c,
Padraig O’Kielyc, Susanne Barthd
a
Leibnitz-Institute for Agricultural Engineering Potsdam-Bornim (ATB), Department of Bioengineering, Max-Eyth-Allee 100, D-
14469 Potsdam, Germany
b
University College Dublin, Department of Industrial Microbiology, Dublin, Ireland
c
Teagasc, Grange Research Centre, Dunsany, Co. Meath, Ireland
d
Teagasc, Oak Park Research Centre, Carlow, Co. Carlow, Ireland

Received 19 April 2005

Abstract

Ensiling plant material with the aid of lactic acid bacteria (LAB) is a common agricultural practice for conserving
forages independently of the time point of harvest. Despite ensiling being a naturally process, it can be improved by the
treatment of the harvested forage with starter cultures before storage. Within this context, Lactobacillus plantarum
(L. plantarum) is the most frequently used LAB in commercially available starter cultures.
In order to enable the monitoring of the population dynamics of L. plantarum in silage, methods for species-specific
detection based on the 16S ribosomal DNA (rDNA) sequence were developed by applying a quantitative
real-time polymerase chain reaction (QRT-PCR) approach. The QRT-PCR assay was also applied to estimate the
development of the L. plantarum population within experimental grass silages. In addition, a multiplex QRT-PCR
assay was developed to estimate the amount of L. plantarum 16S rDNA in relation to total bacterial 16S rDNA.
This multiplex QRT-PCR assay was applied to monitor the influence of different silage additives on the
L. plantarum population.
r 2005 Elsevier GmbH. All rights reserved.

Keywords: Lactobacillus plantarum; Silage; Starter culture; Silage additive; Quantitative real-time PCR; 16S rDNA; TaqMans
probe; 50 -Exonuclease assay

Abbreviations: BLAST, basic local alignment search tool; bp, base
pair(s); cDNA, copy-DNA; CFULAB, colony-forming units of lactic
acid bacteria; C t , threshold cycle; L., Lactobacillus; LAB, lactic acid
bacteria; QRT-PCR, quantitative real-time polymerase chain reaction;
rDNA, ribosomal DNA
Corresponding author.
E-mail address: mklocke@atb-potsdam.de (M. Klocke).
Introduction
The conservation of herbage biomass with the aid of
fermentative bacteria to produce silage for animal
feedstuff is widely used. During ensilage, the anaerobic
microbial conversion of fermentable carbohydrates (i.e.
hexoses and fructose polymers) to organic acids (pre-
dominantly lactic acid) leads to a reduction of the pH

0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.syapm.2005.06.001
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50 M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58
value and thus to a preservation of plant compounds.
An array of anaerobic or facultative-anaerobic micro-
organisms like lactic acid bacteria (LAB), enterobacter-
ia, clostridia and also fungi like yeast can contribute to
this process.
Many additives have been developed which aim to
promote the dominance of the desired LAB
species resulting in a rapid and efficient fermentation
[18,20,34]. Beside molasses, fibrolytic enzymes and
acid-based additives, commonly applied stimulators of
fermentation include inoculants consisting of one or
more LAB strains. A species well suited for such starter
cultures is Lactobacillus plantarum (e.g. [18,35]),
a homofermentative LAB that occurs naturally on
herbage [25].
Despite L. plantarum being a constituent of many
commercially available starter cultures, methods for
monitoring the development of an L. plantarum
population during ensilage are not ideal. Validating
the effectiveness of starter cultures is mainly conducted
indirectly via the estimation of the chemical compounds
produced during ensilage and through assessing the
overall silage quality. One reason for the lack of
adequate monitoring techniques is the absence of clearly
distinguishable phenotypic traits in relation to other
LAB species, which prevents a fast detection via
conventional microbiological techniques like plating or
microscopy.
To overcome these limitations, several approaches
based on the detection of DNA sequences specific for L.
plantarum via polymerase chain reaction (PCR) were
developed. In particularly, two different strategies can
be applied: (i) the analysis of randomly amplified
polymorphic DNA (RAPD) patterns [7,15,21,31,32]
and (ii) the targeting of species-specific sequences in
known genes or DNA regions like the 16S ribosomal
DNA (rDNA) encoding a subunit of the ribosomes
[4,6,15,16] or the intergenic spacer regions between
ribosomal operons [22,28,30].
Currently, the classical endpoint PCR technique is
improved through the development of semi-automated
platforms combined with fluorogenic dyes (SYBRs
Green) or probes (TaqMans probes), which allows
monitoring of product amplification during PCR. Thus,
quantification of the template DNA is possible. Various
quantitative real-time PCR (QRT-PCR) assays based on
the amplification of species-specific sequences within
16S rDNA have been developed in order to detect and
quantify certain bacterial species with medicinal or
economic impact. Applications have been reported
for clinical diagnostics (e.g. [11,19,24]) and for the
detection of contaminants during food processing (e.g.
[3,5,8,33,36]). In this study, we present a first approach
to utilize the QRT-PCR technique for monitoring the
population dynamics of L. plantarum during the ensilage
of grass.
Material and methods
Preparation of a standard
As a reference DNA template for establishing a QRT-
PCR protocol, the plasmid pATB875 containing
approximately 1550 base pairs (bp) PCR amplicon of
the 16S rDNA sequence of L. plantarum was used
(unpublished data). Plasmid DNA was prepared with
the E.Z.N.A. Plasmid Miniprep Kit I (Peqlab Biotech-
nologie, Erlangen, Germany). After purification, the
plasmid DNA was eluted in 0.1
TE buffer [27].
Quantification of plasmid copy number was carried
out via uv-spectrometry according to Sambrook and
Russell [27] and Applied Biosystems [2]. For a standard
plasmid, DNA was diluted with 0.1
TE buffer to
obtain a 10-fold dilution series ranging from 2
109 to
2
104 copies ml1.
Primers and TaqMans probes for QRT-PCR
L. plantarum-specific QRT-PCR primer pair Lplrh-
vreg1-F/-R and the TaqMans probe Lplrh-vreg1-T
(Table 1) were designed with the aid of the Primer
Expresss software (Applied Biosystems, Foster City,
USA) according to the manufacturers guidelines
using standard settings. Lplrh-vreg1-F is based on a
16S rDNA region (Fig. 1) suitable for differentiation
of L. plantarum from other Lactobacillus species [4].
This region was used previously for the development
of species-specific primers for the detection of
L. plantarum in conventional PCR assays [4,16,17].
The TaqMans probe Lplrh-vreg1-T targets a sequence
conserved for most Lactobacillus species within
this variable region. Thus, Lplrh-vreg1-T should
be suitable for application in a broad range of
species-specific QRT-PCR assays. For detection,
Lplrh-vreg1-T was labeled with the fluorescent dyes
VICs (reporter) and TAMRAs (quencher). Lplrh-
vreg1-F/-R were analyzed by nucleotide–nucleotide
basic local alignment search tool (BLAST) [1] against
known bacterial 16S rDNA sequences to avoid false
targeting. The length of the resulting PCR amplicon is
approximately 75 bp.
For detection of the bacterial background the
bacterial universal primers Bac349F and Bac806R
and the TaqMans probe Bac516T were used as
published by Takai and Horikoshi [29]. The target site
of these primers was approximately 170 bp downstream
from the target region of Lplrh-vreg1-F/-R. To be
applicable within multiplex QRT-PCR Bac516T was
labeled with FAMs (reporter) and TAMRAs (quench-
er). The expected PCR amplicon is approximately
450 bp.
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M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58 51

Table 1. Primers for QRT-PCR used in this study

Primer/-sequence Description 3’ Dye 5’ Dye Reference

(reporter) (quencher)

Bac349F Universal forward primer for - - Takai and

3’-AGGCAGCAGTDRGGAAT-5’ bacteria Horikoshi [29]
Bac806R Universal reverse primer - - Takai and
3’-GGACTACYVGGGTATCTAAT-5’ for bacteria Horikoshi [29]
Bac516T Universal TaqMans probe FAMs TAMRAs Takai and
3’-TGCCAGCAGCCGCGGTAATACRDAG-5’ for bacteria Horikoshi [29]
Lplan-vreg1-F Species-specific forward - - this study
3’-TTACATTTGAGTGAGTGGCGAACT-5’ primer for L. plantarum
Lplan-vreg1-R Species-specific reverse - - this study
3’-AGGTGTTATCCCCCGCTTCT-5’ primer for L. plantarum
Lplrh-vreg1-T Universal TaqMans probe VICs TAMRAs this study
3’-GTGAGTAACACGTGGGWAACCTGCCC-5’ for Lactobacillus ssp.

QRT-PCR (50 -exonuclease assay) L. plantarum added as starter culture and (ii) for analysis
of the influence of other silage additives on the total L.
All QRT-PCR reactions were performed on an ABI plantarum population.
7000 system (Applied Biosystems). For estimation of L.
(i) Silage was prepared from chopped grass and
plantarum 16S rDNA copy number the following reaction
inoculated with a starter culture mixture of L.
conditions were optimized according to manufacturers
plantarum ATB 8 and L. rhamnosus ATB 14
guidelines. The reaction mixture in a final volume of 25 ml
[14,17]. Ensiling was carried out in 1.5 l glass jars
H2Odd was: 1
JumpStartTM Taq ReadyMixTM (Sigma-
at the Institute of Agricultural Engineering Bornim.
Aldrich Chemie, Taufkirchen, Germany), 900 nM forward
Comparable replicate silos were opened on 2, 13, 20
primer Lplrh-vreg1-F (MWG Biotech, Ebersberg, Ger-
and 40 days of ensiling and used for further analysis.
many), 300 nM reverse primer Lplrh-vreg1-R (MWG
Chemical analyses of each sample were performed in
Biotech), 200 nM TaqMans probe Lplrh-vreg1-T (Ap-
triplicate. The dry matter (DM) content of fresh
plied Biosystems) and 0.25 ml internal dye ROXs (Sigma-
material and silage was determined by drying for
Aldrich Chemie). The following PCR steps were applied:
48 h at 60 1C followed by 3 h at 105 1C.
(i) 94 1C, 120 s; (ii) 94 1C, 15 s; (iii) 59 1C, 60 s; and (iv)
(ii) Silages were prepared in laboratory silos as described
72 1C, 60 s. Steps (ii)–(iv) were repeated 30 times.
by O’Kiely and Wilson [23] at Teagasc, Grange
For multiplex PCR with parallel detection of L.
Research Centre. Grass was ensiled in triplicate either
plantarum and other bacteria species, the following PCR
as long, unchopped grass or precision-chopped grass
conditions were used modified as described by Takai
after treatment with commercially available silage
and Horikoshi [29] in a final volume of 25 ml H2Odd: 1

additives. Along with an untreated control (variant
JumpStartTM Taq ReadyMixTM, 900 nM Lplrh-vreg1-F,
A) the additives were as follows: (variant B) L.
300 nM Lplrh-vreg1-R, 200 nM Lplrh-vreg1-T, 800 nM
plantarum inoculant based on a mixture of L.
Bac349F (MWG Biotech), 800 nM Bac806R (MWG
plantarum DSM 8862 and L. plantarum DSM 8866
Biotech), 200 nM Bac516T (Applied Biosystems) and
(4.1
1011 CFU t1 silage); (variant C) acid additive
0.25 ml internal dye ROXs. The following cycle regime
based on 85% ammonium tetraformate (3 l t1);
was used: (i) 94 1C, 120 s; (ii) 94 1C, 25 s; and (iii) 57 1C,
(variant D) sucrose (5 kg t1); and (variant E) salt-
360 s. Steps (ii) and (iii) were repeated 30 times.
type additive composed of a mixture of hexamethy-
To reduce experimental setup variation, generally
lene tetra-amine (8%), sodium nitrite (12%), sodium
each QRT-PCR reaction was replicated three times. For
benzoate (15%) and sodium propionate (5%)
each experiment, all QRT-PCR reactions including
(3 l t1). The silos were opened after 110 days and
dilution series of pATB875 as a standard were
aseptically sampled in triplicates. The samples were
performed on one single reaction plate.
stored at 80 1C until further analysis.

Preparation of grass silages

Extraction of bacterial DNA from silage samples
To estimate the applicability of the QRT-PCR assay,
two different types of laboratory scale grass silages were Micro-organisms were isolated before DNA purifica-
analyzed: (i) to monitor the short-term development of tion through washing samples of 4 g silage with 0.1 M
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52 M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58
Fig. 1. Position of the species-specific primers Lpla-vreg1-F/-R and the TaqMans probe Lplrh-vreg1-T on the L. plantarum 16S
rDNA sequence in comparison with the homologous 16S rDNA sequences of other fermentative bacteria commonly found in silage
and Escherichia coli K12. GenBank accession numbers were given in parenthesis after the strain nomination. Asterisks indicate
conserved nucleotides.
sodium phosphate buffer (pH 8.0). Subsequent lysis of
bacterial cells was according to Rheims and Stackeb-
randt [26]. Genomic DNA was further purified with one
phenol:chloroform:isopentylalcohol (25:24:1) step and
two chloroform:isopentylalcohol (24:1) steps [27]. DNA
was precipitated from the aqueous phase by adding
0.25 vol 3 M sodium acetate and 1 vol isopropanol
followed by subsequent incubation at 80 1C overnight.
After centrifugation (10 min at 20,000 rcf) the pellet was
washed twice with 70% ethanol and resuspended in
10 mM Tris/HCl buffer (pH 8.0). Quality and quantity
of genomic DNA were checked via conventional agarose
gel electrophoresis and ethidium bromide staining
[17,27]. For subsequent QRT-PCR analysis DNA
preparations were diluted with 10 mM Tris/HCl buffer
(pH 8.0) from 1:10 to 1:1000.
Estimation of lactic acid bacteria in silage samples
Forty grams subsamples of silage were suspended in
Ringers solution (Merck, VWR International, Darm-
stadt, Germany). From the aqueous phase, 10-fold serial
dilution series were made in Ringers solution. A 100 ml
aliquot from each dilution was plated on MRS agar
plates (Merck), followed by incubation at 30 1C for 2
days. From suitable dilutions all grown colonies were
counted visually and used for calculating the number of
colony-forming units of LAB (CFULAB) within the
silage.
Quantification of organic acid content in silage
samples
Acetic acid, propionic acid, butyric acid and isovaleric
acid in silage samples were quantified with gas
chromatography on a Fisons GC 8000 system (Fisons
Instruments, Mainz, Germany) according to the proto-
col published by Idler [13] with a minor modification
(split setting at 1:20). Additionally, lactic acid was
quantified with HPLC analysis using a cold toluene
extract for the extraction of lactic acid. HPLC analysis
was carried out on a Dionex-System (Dionex, Idstein,
Germany) using a Eurokat H ion exchange column
(column size 300
8 mm, particle size 10 mm) (Knauer,
Berlin, Germany) in combination with a Shodex 71
refraction index detector (Dionex). In all, 0.01 M H2SO4
was used as mobile phase. Operation settings were:
pressure 80 bar, column temperature 35 1C, flow rate
0.8 ml min1 and injection volume 10 ml. The quantified
concentrations of all acids were combined to obtain the
total organic acid content within silage samples.
Results
Development of a QRT-PCR assay (50 -exonuclease
assay) for L. plantarum
A QRT-PCR primer set including the L. plantarum-
specific primer pair Lplrh-vreg1-F and Lplrh-vreg1-R
and the TaqMans probe Lplrh-vreg1-T (Table 1, Fig. 1)
was developed in silico. Subsequently, this primer set
was tested using different amounts of plasmid pATB875
containing a 1550 bp amplicon of L. plantarum 16S
rDNA as template. The amplification of 16S rDNA was
linear within the range of 104–1010 copies (Fig. 2A and
B). No unspecific PCR products were amplified as
verified by gel electrophoresis of the PCR products. For
all subsequent experiments, aliquots of this dilution
series (106 up to 109 copies of pATB875 per reaction)
were used as a standard on each PCR reaction plate.
Similar amplification ratios were obtained combining
Lplrh-vreg1-F/-R and Lplrh-vreg1-T with the bacterial
universal primers Bac349F, Bac806R and the TaqMans
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M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58 53

Fig. 2. Representative QRT-PCR amplification curves (A, C) and resulting threshold cycle values (C t ) (B, D) using pATB875 as
template. (A, B) QRT-PCR assay applying Lplrh-vreg1-F/-R and Lplrh-vreg1-T; (C, D) multiplex QRT-PCR assay applying Lplrh-
vreg1-F/-R and Lplrh-vreg1-T in combination with universal primers for detection of bacterial 16S rDNA. (J) 1
1010, (W)
1
109, (&) 1
108, (K) 1
107, (m) 1
106, (’) 1
105, copies of pATB875.
¼ Control reaction without DNA template.
Horizontal lines represent the values for the threshold cycle (C t ). Error bars indicate standard deviation of three replicates:
r2 ¼ 0:9988 in (B) and 0.9968 in (D).

probe Bac516T using pATB875 as template (Fig. 2C
and D). The exponential dependency of amplification of
L. plantarum 16S rDNA in the presence of other PCR
primers was determined for a range of 105 up to 1010
copies.
Using L. rhamnosus DNA as a non-L. plantarum
DNA background, the multiplex QRT-PCR assay was
able to detect differences within concentrations of L.
plantarum 16S rDNA ranging from approximately 108
to 105 copies (Fig. 3). The threshold cycle value was
exponential to the amount of L. plantarum independent
of the simultaneous application of the L. plantarum-
specific primer pair Lplrh-vreg1-F/-R and the bacterial
universal primer pair Bac349F and Bac806R (Fig. 4).
Applying a multiplex approach, the universal bacter-
ial primer Bac349F and Bac806R also detected the total
amount of bacterial DNA (in this case, L. plantarum and
L. rhamnosus DNA). A two-fold alteration of genomic
DNA was detected by an altered C t value; minor
alterations of the total DNA concentrations were not
recognized. In contrast to Lplrh-vreg1-F/-R, the ampli-
fication curves derived from Bac349F and Bac806R
showed a reproducible lower plateau phase in the
presence of higher amounts of L. plantarum DNA
(Fig. 3).
QRT-PCR-based analysis of experimental grass
silages
The L. plantarum-specific QRT-PCR assay was
applied to monitor the dynamics of an L. plantarum
population within grass silages inoculated with a starter
culture consisting of equal concentrations of L. plantar-
um ATB 8 and L. rhamnosus ATB 14. For this propose,
DNA was prepared from similar amounts of silage
taken at different time points during ensilage. Amplifi-
cation curves for Lplrh-vreg1-F/-R showed reproducible
differences between the amount of L. plantarum 16S
rDNA within the DNA preparations: the C t values
decreased from 24.4 (70.4) at day 2 to 21.9 (70.4) on
day 13 indicating an increase of the L. plantarum
population. During continued ensiling the C t values
increased again to 23.6 (70.1) at day 20 and 25.7 (70.2)
at day 40.
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54 M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58

Fig. 3. Product amplification curves of multiplex QRT-PCR using different amounts of genomic DNA of L. plantarum and L.
rhamnosus ( ¼ non-L. plantarum background). DNA concentration ratios for L. plantarum and L. rhamnosus DNA were 1:1 (K),
1:10 (m), 1:100 (’) and 1:1000 (J).
¼ Control reaction without DNA template. Error bars indicate standard deviation of three
replicates. Horizontal lines indicate threshold cycle C t (automatic set value).

total organic acid contents in the silage samples

Fig. 4. Threshold cycle (C t ) values for amplification of L.
plantarum 16S rDNA in multiplex QRT-PCR (solid lines,
circles) and in non-multiplexed QRT-PCR (dashed lines,
triangles) in the presence of non-L. plantarum DNA. Error
bars indicate standard deviation of three replicates
(r2 ¼ 0:9996 for multiplex and 0.9989 for non-multiplexed
QRT-PCR).
The subsequent calculated values for L. plantarum
16S rDNA copy number indicated an increase of L.
plantarum within the silage during the first 2 weeks
followed by a decrease during the following weeks
(Fig. 5). This result corresponds well with the popula-
tion changes of total LAB as estimated by CFULAB
counts except at day 40. At this time, the CFULAB
counts reveal 1.4
106 LAB per gram fresh matter,
approximately 1000-fold less than the calculated values
for L. plantarum 16S rDNA. These population dynamics
were also supported by the analysis of the lactic acid and
displaying a continuous accumulation of these acids.
Because of variability in the efficiency of the DNA
extraction process, the QRT-PCR assay could be
influenced by variable DNA concentrations within the
DNA preparations. To avoid this fundamental problem,
an alternative approach using a multiplex QRT-PCR
assay was applied. The detection of total bacterial 16S
rDNA parallel to L. plantarum 16S rDNA should be
used for the estimation of the relative amount of L.
plantarum within the total bacterial microflora. Thus, a
multiplex QRT-PCR was carried out on DNA prepared
from different pretreated silage samples (Fig. 6).
The multiplex QRT-PCR assay revealed reproducible
differences between the different silage additives (Fig. 7).
DNA preparations derived from silage treated with a
commercially available starter culture consisting of two
different L. plantarum strains showed an increased value
of L. plantarum 16S rDNA in relation to the value of
total bacterial 16S rDNA compared to the untreated
sample as indicated through the DC t values (Fig. 7A and
B). These differences were not only detectable by
differences in the C t values, but were also detectable in
the different amplification curves especially during the
plateau phase (Fig. 6).
The DC t values of experiments with DNA prepared
from silage with a different additive treatment showed
also differences to the untreated sample (Fig. 7C–E).
However, these differences were in a smaller scale than
the case of silage treated with an L. plantarum inoculant.
Also differences between due to chopping the forage
were observed. Except the sample treated with starter
cultures, silage made from grass that was precision
chopped showed lower DC t values indicating higher
amounts of L. plantarum within the bacterial microflora.
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M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58 55

Fig. 5. Estimation of L. plantarum 16S rDNA copy number, CFU of LAB, and organic acid content in grass silages pretreated with
L. plantarum ATB 8 and L. rhamnosus ATB 14. Error bars indicate standard deviation of three replicates (for 16S rDNA copy
number only). The asterisk indicates a value of 1.4
106 CFULAB bacteria for day 40.

Fig. 6. Representative multiplex QRT-PCR amplification

Fig. 7. Results of a multiplex QRT-PCR experiment of
curves applying universal bacterial primers (dashed lines, open
various grass silage samples applying universal bacterial
symbols) and primers specific for L. plantarum (solid lines,
primers and primers specific for L. plantarum. The DC t value
solid symbols). Samples were taken from silages pretreated
was used to estimate differences in the amount of targeted 16S
with starter cultures containing L. plantarum strains (m, W)
rDNA for total bacteria and L. plantarum depending on the
and silages without any additives (K, J). Horizontal bar
type of silage treatment. The following silage additives were
indicates the value for the threshold cycle (C t ). Error bars
applied: (A) no additive; (B) L. plantarum inoculant based on a
indicate standard deviation of three replicates.
mixture of L. plantarum DSM 8862 and L. plantarum DSM
8866 (4.1
1011 CFU t1 silage); (C) acid-based additive; (D)
sucrose; (E) salt-type additive.

Discussion
In recent times, 16S rDNA-based PCR assays have
been proven to be a valuable addition to classical
microbiological techniques for the identification of
bacteria. They allow a species-specific detection of
bacteria even in difficult substrates (e.g. environmental
samples) without time-consuming cultivation steps.
Furthermore, the introduction of fluorogenic dyes or
probes combined with automated detection enables a
precise estimation of the amount of starting DNA
template. Many examples for the application of QRT-
PCR assays were published for the detection of certain
bacterial species of clinical or economic relevance.
Besides assays for the detection of pathogenic bacteria,
methods were developed mainly for the detection of
contaminants which interfere with food production
processes [3,5,8,33,36]. However, to our knowledge, no
applications of this powerful technique for the analysis
of non-food fermentations like ensilage have been
published so far.
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56 M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58
In this study, a first QRT-PCR-based approach is
presented for monitoring one bacterial species, L.
plantarum, commonly applied as silage additive. For
this approach, the QRT-PCR primer Lplrh-vreg1-F was
developed in silico targeting an L. plantarum-specific 16S
rDNA region (according to 87–111 bp Escherichia coli
K12 16S rDNA) (Fig. 1). This region proved to be a
suitable target for the development of L. plantarum-
specific PCR primers in earlier works carried out by
Chagnaud et al. [4] and Klocke and Mundt [17]. In
combination with the newly developed reverse
primer Lplrh-vreg1-R, the QRT-PCR primer pair
presented in this study showed their ability to distin-
guish L. plantarum from the comparatively closely
related L. rhamnosus (Fig. 3). Nevertheless, due to the
high diversity of LAB found in silage [10], it cannot be
excluded that the QRT-PCR primer pair Lplrh-vreg1-
F/-R used in this study also targets other LAB species.
The QRT-PCR assay was applied for monitoring the
behavior of starter cultures during ensiling. At different
time points of silage fermentation this QRT-PCR assay
revealed a similar population development for L.
plantarum as for total LAB estimated by CFULAB
analysis (Fig. 5). However, this approach is still
insufficient because a quantification of L. plantarum is
not possible without further calibration experiments.
Especially during prolonged ensiling (20 and 40 days),
larger derivations between the 16S rDNA copy number
as indicated by the QRT-PCR assay and the CFULAB
analysis were observed. Beside the detection of multiple
16S rDNA loci within one single L. plantarum genome
by QRT-PCR, these derivations may be also introduced
by the simultaneous detection of living and dead cells by
the QRT-PCR assay. As shown by Hupfer et al. [12],
shorter DNA fragments (up to 226 bp) were detectable
by PCR up to 100 days of ensiling. Thus, the application
of RNA (respectively, the reverse-transcribed copy-
DNA (cDNA)) as target for the subsequent QRT-PCR
may lead to a further improvement of the assay.
Another fundamental problem is the variability of the
QRT-PCR introduced through variations during pre-
paration and purification of genomic DNA and during
QRT-PCR setup [9]. In addition to the analysis of
replicates, a suitable strategy to reduce this variability
would be the inclusion of a reference. For this purpose,
the amount of total bacterial 16S rDNA was estimated
according to Takai and Horikoshi [29]. Both assays were
successfully combined in one multiplex assay. The
presence of a second primer pair did not influence the
C t value, thus an estimation of 16S rDNA copy number
was feasible. Interestingly, depending on the amount of
L. plantarum DNA, an altered plateau phase of the
amplification curve for the bacterial 16S rDNA was
observed combined with a low reproducibility during
late plateau phase (Fig. 3B and 6). This findings may be
caused by the large differences in size between the L.
plantarum-specific amplicon (approximately 75 bp) and
the total bacteria amplicon (approximately 450 bp) and,
thus, by possible different optimal reaction conditions.
Potentially an optimized primer-design for detection of
total bacterial 16S rDNA as an alternative to the
previously published primers [29] used in this study
would reduce this variability. However, the results
presented in Fig. 4 clearly indicate that the C t values
as characteristic parameters for the amount of initial
16S rDNA were not affected by the multiplexed primers.
Additionally, the multiplex QRT-PCR assay was also
applied to the analysis of grass silage samples treated
with different additives at ensiling (Fig. 7). As expected,
the assay showed the highest L. plantarum 16S rDNA/
total bacterial 16S rDNA ratio in silage with starter
cultures consisting of L. plantarum as silage additive.
Other additives, like acid-based additives, seem to
support the L. plantarum population in relation to
starter cultures in a lower scale as indicated through the
higher DC t values. Thus, this multiplex QRT-PCR is a
suitable tool for estimating of the effectiveness of silage
additives concerning their influence on the L. plantarum
population. However, it should be noted that our
experiments are a first attempt in marker-based analysis
of silage additives reflecting only single silage samples.
For a reliable estimation of the development of L.
plantarum during ensiling, greater replication is re-
quired.
This study showed for the first time that the QRT-
PCR method is applicable for direct monitoring of
selected fermentative bacteria in silage. The QRT-PCR
assay presented in this study could be a more specific
alternative to the classical MRS-agar-plate-based count-
ing of CFU with the potential of a better automatization
for routine analysis. Further investigations are necessary
to enable estimation of the number of living cells and to
provide a more robust QRT-PCR assay. Ideally, a set of
species-specific QRT-PCR assays should be developed
to provide a tool for fast and reliable analysis of
microbial dynamics during ensiling.
Acknowledgements
This work was supported in part by the European
Molecular Biology Organization (EMBO), Heidelberg,
Germany (ASTF 220-04). The authors are grateful to
G. Carlow, U. Knuth and G. Rehde (ATB) for their
valuable technical support.
References
[1] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J.
Lipman, Basic local alignment search tool, J. Mol. Biol.
215 (1990) 403–410.
 ARTICLE IN PRESS
M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58
[2] Applied Biosystems, Creating standard curves with
genomic DNA or plasmid DNA templates for use in
quantitative PCR, published online at URL: http://
www.appliedbiosystems.com/support/tutorials/pdf/
quant_pcr.pdf,2003.
[3] A.A. Bhagwat, Simultaneous detection of Escherichia coli
O157:H7, Listeria monocytogenes and Salmonella strains
by real-time PCR, Int. J. Food Microbiol. 84 (2003)
217–224.
[4] P. Chagnaud, K. Machinis, L.A. Coutte, A. Marecat,
A. Mercenier, Rapid PCR-based procedure to identify
lactic acid bacteria: application to six common Lactoba-
cillus species, J. Microbiol. Meth. 44 (2001) 139–148.
[5] Z. Cheng, M.W. Griffiths, Rapid detection of Campylo-
bacter jejuni in chicken rinse water by melting-peak
analysis of amplicons in real-time polymerase chain
reaction, J. Food Prot. 66 (2003) 1343–1352.
[6] M.D. Collins, U. Rodrigues, C. Ash, M. Aguirre, J.A.E.
Farrow, A. Martinez-Murcia, B.A. Phillips, A.M. Wil-
liams, S. Wallbanks, Phylogenetic analysis of the genus
Lactobacillus and related lactic acid bacteria as deter-
mined by reverse transcriptase sequencing of 16S rRNA,
FEMS Microbiol. Lett. 77 (1991) 5–12.
[7] S.M. Cusick, D.J. O’Sullivan, Use of a single, triplicate
arbitrarily primed-PCR procedure for molecular finger-
printing of lactic acid bacteria, Appl. Environ. Microbiol.
66 (2000) 2227–2231.
[8] A. Delaherche, O. Claisse, A. Lonvaud-Funel, Detection
and quantification of Brettanomyces bruxellensis and
‘ropy’ Pediococcus damnosus strains in wine by real-time
polymerase chain reaction, J. Appl. Microbiol. 97 (2004)
910–915.
[9] H.M. Dionisi, G. Harms, A.C. Layton, I.R. Gregory,
J. Parker, S.A. Hawkins, K.G. Robinson, G.S. Sayler,
Power analysis for real-time PCR quantification of genes
in activated sludge and analysis of the variability
introduced by DNA extraction, Appl. Environ. Micro-
biol. 69 (2003) 6597–6604.
[10] S. Ennahar, Y.M. Cai, Y. Fujita, Phylogenetic diversity of
lactic acid bacteria associated with paddy rice silage as
determined by 16S ribosomal DNA analysis, Appl.
Environ. Microbiol. 69 (2003) 444–451.
[11] O. Greiner, P.J.R. Day, P.P. Bosshard, F. Imeri,
M. Altwegg, D. Nadal, Quantitative detection of
Streptococcus pneumonie in nasopharyngal secretions
by real-time PCR, J. Clin. Microbiol. 39 (2001)
3129–3134.
[12] C. Hupfer, J. Mayer, H. Hotzel, K. Sachse, K.-H. Engel,
The effect of ensiling on PCR-based detection of
genetically modified Bt maize, Eur. Food Res. Technol.
209 (1999) 301–304.
[13] C. Idler, Ernte, Konservierung und Erstverarbeitung von
Hanf aus einer Feuchtgutlinie, Forschungsberichte ATB
2000/03, Institute of Agricultural Engineering Bornim,
Potsdam, 2000.
[14] C. Idler, K. Richter, R. Partzsch, G. Neubert, F. Heber,
Homemade starter cultures for silage, Landtechnik 57
(2002) 261–263.
[15] M.L. Johansson, M. Quednau, G. Molin, S. Ahrne,
Randomly amplified polymorphic DNA (RAPD) for
57
rapid typing of Lactobacillus plantarum strains, Lett.
Appl. Microbiol. 21 (1995) 155–159.
[16] M. Klocke, A. Knapik, K. Mundt, Den Bakterien auf der
Spur: Marker-gestützte Detektion von Starterkulturen für
die Grünfutter-Silierung, Agrartech. Forsch. 10 (2004)
98–104.
[17] M. Klocke, K. Mundt, Development of a 16S rDNA-
targeted PCR assay for monitoring of Lactobacillus
plantarum and Lact. rhamnosus during co-cultivation for
production of inoculants for silages, Lett. Appl. Micro-
biol. 39 (2004) 267–273.
[18] L. Kung, M.R. Stokes, C.J. Lin, Silage additives, In: D.R.
Buxton, R.E. Muck, J.H. Harrision (Eds.), Silage Science
and Technology, American Society of Agronomy, Madi-
son, 2003, pp. 305–360.
[19] S.H. Lyons, A.L. Griffen, E.J. Leys, Quantitative real-
time PCR for Porphyromonas gingivalis and total
bacteria, J. Clin. Microbiol. 38 (2000) 2362–2365.
[20] P. McDonald, A.R. Henderson, S.J.E. Heron, The
Biochemistry of Silage, second ed., Chalcombe Publica-
tions, Lincoln, 1991.
[21] A. Nigatu, S. Ahrne, G. Molin, Randomly amplified
polymorphic DNA (RAPD) profiles for the distinction of
Lactobacillus species, Antonie Van Leeuwenhoek Int.
J. Gen. Mol. Microbiol. 79 (2001) 1–6.
[22] M. Nour, 16S–23S and 23S–5S intergenic spacer regions
of lactobacilli, nucleotide sequence, secondary structure
and comparative analysis, Res. Microbiol. 149 (1998)
433–448.
[23] P. O’Kiely, R.K. Wilson, Comparison of three silo types
used to study in-silo processes, Ir. J. Agric. Res. 30 (1991)
53–60.
[24] A. Pahl, U. Kühlbrandt, K. Brune, M. Röllinghoff,
A. Gessner, Quantitative detection of Borrelia burgdorferi
by real-time PCR, J. Clin. Microbiol. 37 (1999)
1958–1963.
[25] G. Pahlow, R.E. Muck, F. Driehus, S.J.W.H. Oude
Elferink, S.F. Spoelstra, Microbiology of ensiling, In:
D.R. Buxton, R.E. Muck, J.H. Harrision (Eds.), Silage
Science and Technology, American Society of Agronomy,
Madison, 2003, pp. 31–93.
[26] H. Rheims, E. Stackebrandt, Application of nested
polymerase chain reaction for the detection of as yet
uncultured organisms of the class Actinobacteria in
environmental samples, Environ. Microbiol. 1 (1999)
137–143.
[27] J. Sambrook, D.W. Russell, Molecular Cloning – A
Laboratory Manual, third ed., Cold Spring Harbour
Laboratory Press, Cold Spring Harbour, 2001.
[28] Y.-L. Song, N. Kato, C.-X. Liu, Y. Matsumiya, H. Kato,
K. Watanabe, Rapid identification of 11 human intestinal
Lactobacillus species by multiplex PCR assays
using group- and species-specific primers derived from
the 16S–23S rRNA intergenic spacer region and its
flanking 23S rRNA, FEMS Microbiol. Lett. 187 (2000)
167–173.
[29] K. Takai, K. Horikoshi, Rapid detection and quantifica-
tion of members of the archaeal community by quanti-
tative PCR using fluorogenic probes, Appl. Environ.
Microbiol. 66 (2000) 5066–5072.
 ARTICLE IN PRESS
58 M. Klocke et al. / Systematic and Applied Microbiology 29 (2006) 49–58
[30] G.W. Tannock, A. Tilsala-Timisjarvi, S. Rodtong, J. Ng,
K. Munro, T. Alatossava, Identification of Lactobacillus
isolates from the gastrointestinal tract, silage, and yoghurt
by 16S–23S rRNA gene intergenic spacer region sequence
comparisons, Appl. Environ. Microbiol. 65 (1999)
4264–4267.
[31] A. Tilsala-Timisjarvi, T. Alatossava, Strain-specific
identification of probiotic Lactobacillus rhamnosus
with randomly amplified polymorphic DNA-derived
PCR primers, Appl. Environ. Microbiol. 64 (1998)
4816–4819.
[32] S.Z. Validov, N.V. Pankova, E.V. Kozlova, N.P.
Kuzmin, V.V. Klimenko, A.M. Boronin, Rapid identifi-
cation of Lactobacillus plantarum by RAPD-PCR, Micro-
biology 67 (1998) 317–322.
[33] J.S. Van Kessel, J.S. Karns, M.L. Perdue, Using a
portable real-time PCR assay to detect Salmonella in
raw milk, J. Food Prot. 66 (2003) 1762–1767.
[34] Z.G. Weinberg, R.E. Muck, New trends and opportu-
nities in the development and use of inoculants for silage,
FEMS Microbiol. Rev. 19 (1996) 53–68.
[35] A.G. Whiter, L. Kung, The effect of a dry or liquid
application of Lactobacillus plantarum MTD1 on the
fermentation of alfalfa silage, J. Dairy Sci. 84 (2001)
2195–2202.
[36] C. Yang, Y. Jiang, K. Huang, C. Zhu, Y. Yin,
Application of real-time PCR for quantitative detection
of Campylobacter jejuni in poultry, milk and environ-
mental water, FEMS Immunol. Med. Microbiol. 38
(2003) 265–271.