Download Specificity of a Polymerase Chain Reaction Assay of a Target

Eur J Clin Microbiol Infect Dis (2001) 20 : 127–131
Q Springer-Verlag 2001
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Specificity of a Polymerase Chain Reaction Assay of a
Target Sequence on the 31-Kilodalton Brucella Antigen
DNA Used to Diagnose Human Brucellosis
M.C. Casañas, M.I. Queipo-Ortuño, A. Rodriguez-Torres, A. Orduña, J.D. Colmenero,
P. Morata
Abstract The aim of this study was to evaluate the
specificity of a polymerase chain reaction assay for
detecting Brucella DNA using primers specific for the
amplification of a 223 bp region of the sequence
encoding a 31 kDa immunogenic Brucella abortus
protein (BCSP31). DNA from all Brucella strains,
including type, reference, vaccine and field strains,
were correctly amplified. With the exception of Ochrobactrum spp., no other amplification was detected with
a broad panel of microorganisms serologically or phylogenetically related to Brucella spp. This very good
degree of specificity, together with its high yield
demonstrated in previous clinical studies, confirms that
this polymerase chain reaction assay could be a useful
tool for the diagnosis of human brucellosis.
Introduction
Brucellosis is a zoonosis that produces severe morbidity
in humans [1]. The disease exists worldwide and is
endemic in some Mediterranean countries, where it
represents an important public health problem.
Because the clinical picture of brucellosis is nonspecific
M.C. Casañas
Microbiology Service, “Carlos Haya” Hospital, Malaga, Spain
M.I. Queipo-Ortuño, P. Morata
Department of Biochemistry and Molecular Biology,
Faculty of Medicine, University of Malaga, Spain
A. Rodriguez-Torres, A. Orduña
Department of Microbiology, University Hospital,
Faculty of Medicine, Valladolid, Spain
J.D. Colmenero (Y)
Infectious Diseases Unit, Department of Internal Medicine,
“Carlos Haya” Hospital, Camino de Antequera s/n,
29010 Malaga, Spain
e-mail: colmene6interbook.net
Tel.: c34-95-2645809
Fax: c34-95-2288349
and may show great variability, its diagnosis requires
laboratory confirmation [2]. However, the laboratory
diagnosis of brucellosis is hampered by the slow growth
of Brucella spp. in culture and its danger to laboratory
personnel [3]. Moreover, although serological tests are
easy to perform, they suffer from a lack of specificity,
which makes accurate detection of the illness particularly difficult in patients with a recent history of brucellosis, in patients with a suspected relapse, and in areas
where the disease is endemic [4].
To date, few attempts to apply techniques of molecular
biology to the diagnosis of human brucellosis have
been made. Some studies have detected small amounts
of Brucella DNA in pure cultures and animal samples
by means of polymerase chain reaction (PCR) [5, 6].
Our group recently developed a one-step PCR technique consisting of the amplification of a fragment of
223 base pairs of an antigenic protein of Brucella
abortus of 31 kDa (BCSP31) using the B4 and B5
primers previously described by Baily et al. [7]. Those
investigators tested the specificity of these primers in
DNA extracted from Branhamella catarrhalis, Yersinia
enterocolitica, Campylobacter jejuni, Enterobacter aerogenes, Haemophilus influenzae, Legionella pneumophila and Escherichia coli; no DNA amplification was
detected. On the basis of Baily’s results concerning
primer specificity, we concentrated on optimizing the
PCR technique for its use in peripheral blood samples
and its diagnostic yield compared with microbiological
tests conventionally used for the diagnosis of brucellosis, for post-treatment control and for early detection
of relapses [8, 9].
Although the technique has been highly sensitive and
specific in clinical studies [8, 9], the existence of the
occasional false-positive result prompted us to carry
out this study. Our aim was to verify the specificity of
the technique using a wide panel of microorganisms
serologically or phylogenetically related to Brucella
128
spp. and strains from patients with clinical pictures
involving a differential diagnosis with brucellosis.
pellet was redissolved in 0.1 ml of sterile Milli-Q water and rehydrated overnight at room temperature.
Materials and Methods
For both methods, the concentration and purity of the DNA were
determined spectrophotometrically by readings at A260/A280 nm.
Samples were diluted up to 100 ng/ml and stored at 4 7C until use.
After the DNA extraction, purification and measurement steps,
most authors store it at –20 7C. However, throughout all our
studies we have observed that when DNA is to be used as a
sample for PCR, its yield is greater when stored at 4 7C than at
–20 7C, even with prolonged storage times (8).
Bacterial Strains and Growth Conditions. The strains of Brucella
spp. used in this study that are listed in Table 1 were provided by
the Microbiology Department of the Faculty of Medicine at
Valladolid University. In earlier studies, we used the vaccine
strains B-19 and Rev-1 as positive controls; these strains were
generously provided by the Agriculture Department of the Andalusian Regional Government [8, 9]. These strains were cultured
on Brucella agar (Difco, USA) and incubated at 37 7C with 5%
CO2 for 48 h. Other bacteria, both with and without phylogenetic
or antigenic relation to Brucella spp., were also tested (Tables 1
and 2). Neisseria meningitidis, Haemophilus influenzae and Francisella tularensis were cultured at 37 7C on chocolate agar with
10% CO2 for 24–48 h. Agrobacterium spp., Phyllobacterium spp.
and Ochrobactrum anthropi were grown on blood agar (Difco) at
25 7C for 48 h and Yersinia enterocolitica on the same medium at
30 7C for 24 h. The strains of Mycobacterium tuberculosis were
grown on Löwenstein-Jensen medium (Biomedics, Spain) at 37 7C
for 3–4 weeks. Because it grows so slowly, the lyophilized Bartonella bacilliformis strain supplied by the Pasteur Institute Paris,
France, was processed directly after reconstituting it in phosphate-buffered saline (PBS). Other bacteria used in this study
were grown on blood agar (Difco) at 37 7C for 24 h. All organisms
were harvested in NaCl 0.85% and the turbidity of bacterial
suspensions was adjusted to a McFarland 1–3 standard. Brucella
spp., Listeria monocytogenes, Francisella tularensis, Neisseria
meningitidis, Salmonella spp. and Mycobacterium tuberculosis
were killed with equal parts of pure acetone and NaCl 0.85% held
in suspension at 40 7C overnight.
Preparation of Genomic DNA. We used two methods of DNA
extraction: salting out, as described by Miller et al. [10] and
modified by us [8], and the standard method with phenol/chloroform/isoamyl alcohol for gram-positive bacteria and fungi.
Extraction of Genomic DNA from Bacterial Cultures using the
Salting-Out Method. This method involves salting out the
cellular proteins obtained from the cellular lysate with proteinase
K by dehydration and precipitation with a saturated (7.5 M)
ammonium acetate solution. Bacterial cells were washed twice
with PBS and pelleted by centrifugation. The pellet of gram-negative bacteria was resuspended in a solution containing 68 ml of
20 mg/ml lysozyme, 40 ml of 10% sodium dodecyl sulfate (SDS),
80 ml of lysis buffer (375 mM NH4Cl, 120 mM Na2-EDTA [pH
8.0]) and 157 ml of sterile Milli-Q water, then mixed and incubated for 30 min at 37 7C. For gram-positive bacteria and fungi,
the solution contained 100 ml of 20 mg/ml lysozyme, 40 ml of 10%
SDS, 80 ml of lysis buffer and 125 ml of sterile Milli-Q water. The
incubation was performed for 1 h at 37 7C. Then, 40 ml of 10 mg/
ml proteinase K was added to each tube for both gram-negative
and gram-positive bacteria, mixed gently by inverting the tubes
several times and incubated for 30 min at 55 7C. Purification and
precipitation of DNA was performed as previously reported [8].
Extraction of Genomic DNA from Gram-Positive Bacteria using
the Phenol-Chloroform Method. Gram-positive bacteria suspensions were washed twice with PBS, pelleted by centrifugation,
resuspended in 0.5 ml of TE buffer (10 mM Tris-HCl, 1 mM
EDTA) and incubated at 37 7C for 1 h with 0.5% SDS and proteinase K (100 mg/ml). Cell-wall debris, polysaccharides and the
remaining proteins were removed by selective precipitation with
7.5 M ammonium acetate and CTAB-NaCl solution and incubated at 65 7C for 10 min. DNA was extracted using a standard
protocol with phenol-chloroform-isoamyl alcohol, precipitated
with isopropanol, washed with 70% ethanol and dried. The DNA
DNA Amplification. This procedure was performed as we have
previously described [8]. The primers B4 (5b tggctcggttgccaatatcaa
3b) and B5 (5b cgcgcttgcctttcaggtctg 3b) [7] generated a 223 bp
product of the conserved region of the gene (nucleotides
789–1012), which encodes a protein of 31 kDa Brucella abortus
antigen. PCR was performed in a 50-ml mixture containing 100 ng
template DNA, PCR buffer [10 mMTris-HCL (pH 8.4), 50 mM
KCL, 1.0 mM MgCl2], 100 nM of each PCR primer (Pharmacia
LKB, Barcelona, Spain), 200 mM each deoxyribonucleoside
triphosphate (dNTPs; Boehringer Mannheim, Germany) and
1.25 U Taq polymerase (Boehringer Mannheim). The reaction
was performed in a DNA thermal cycler without mineral oil
(Perkin-Elmer 2400; USA.). PCR consisted of preheating at 93 7C
for 5 min, 35 cycles of 90 7C for 1 min, 60 7C for 30 s and 72 7C for
1 min, and incubation at 72 7C for 7 min. The PCR products were
electrophoretically separated on agarose gel (2%) and stained
with 2 mg/ml ethidium bromide to determine the size of the
amplified products.
Negative controls of PCR containing all of the reagents but
lacking template DNA were routinely processed exactly as has
been described to monitor for contamination with Brucella DNA.
All were negative in all experiments. Positive controls with 100 ng
of genomic DNA isolated from a suspension of Brucella abortus
B-19 were included in each experiment. All PCR reactions were
carried out in duplicate. A strain of Escherichia coli from our
hospital was used as a control for the DNA extraction and
contamination: its PCR was negative in all the experiments
carried out.
Results and Discussion
The extraction and purification of DNA in our
previous studies was made by salting out, with excellent
yields in concentration and purity. We therefore
decided to apply the same technique to all the microorganisms tested in the present study. A good yield was
obtained for gram-negative bacteria for both concentration and purity with the salting-out method,
although there was considerable variability in the
amount of DNA for the different strains. With regard
to the various Brucella strains, values of DNA above
100 mg/ml were obtained in all cases with a high degree
of purity. On the other hand, the DNA obtained from
the gram-positive microorganisms with the salting-out
method was much less than that for the gram-negative
organisms, even though we modified the original procedure by using higher concentrations of lysozyme and a
longer incubation period. However, DNA extraction of
gram-positive microorganisms with the phenol-chloroform method increased the yield considerably, although
the purity was substantially reduced (data not shown).
129
Table 1 PCR results with DNA from different Brucella strains and from bacteria antigenically or genetically related to Brucella
spp.
Species (no. of isolates)
Biovar
Strain
Origin
PCR result
Brucella melitensis
Brucella melitensis
Brucella melitensis
Brucella melitensis
Brucella melitensis (5)
Brucella melitensis (6)
Brucella abortus (2)
Brucella abortus
Brucella abortus
Brucella abortus
Brucella abortus
Brucella abortus
Brucella abortus
Brucella abortus
Brucella abortus
Brucella suis
Brucella suis
Brucella suis
Brucella suis
Brucella suis
Brucella neotomae
Brucella ovis
Brucella canis
Antigenically related bacteria
Escherichia coli
Francisella tularensis
Pasteurella multocida
Salmonella urbana
Yersinia enterocolitica
Genetically related bacteria
Agrobacterium radiobacter
Agrobacterium tumefaciens
Agrobacterium vitis
Bartonella bacilliformis
Ochrobactrum intermedium
Ochrobactrum anthropi
Ochrobactrum anthropi
Ochrobactrum anthropi
Phyllobacterium myrsincearum
Phyllobacterium rubiacearum
Vibrio cholerae
1
1
2
3
2
3
1
1
2
3
4
5
6
7
9
1
2
3
4
5
16 M
Rev 1
63/9
Ether
FMV
CAJA
FMV
FMV
FMV (clinical strain)
FMV (clinical strain)
FMV (clinical strain)
CAJA
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
FMV
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
CECT
FMV
CECT
CECT
FFG
P
P
P
P
P
CECT
CECT
CECT
IP
FMN
FMN
CECT
HCH
CECT
CECT
CECT
P
P
P
P
c
c
c
P
P
P
P
B19
86/8/59
Tulya
292
B3196
870
63/75
C/68
10036
10510
10511
40
10980
10084
Reo198
10854
O:157
H: 7
O:9
3301
3331
FMV, Facultad de Medicina Valladolid, Valladolid, Spain;
CAJA, Consejeria de Agricultura, Junta de Andalucia, Seville,
Spain; CECT, Colección Española de Cultivos Tipo, Valencia,
Spain; FFG, Facultad de Farmacia de Granada, Granada, Spain;
FMN, Facultad de Medicina de Navarra, Pamplona, Spain; IP,
Institut Pasteur, Paris, France; HCH, Hospital Carlos Haya,
Málaga, Spain
All strains of Brucella spp. showed clear amplification
of the DNA with the PCR we developed (Table 1).
These results, therefore, support those obtained
previously by DNA-DNA hybridization and different
PCR assays using primers specific for the genes
encoding different Brucella proteins and 16S rRNA [11,
12].
are able to produce a clinical picture involved in the
differential diagnosis of brucellosis (Table 2).
With the exception of Ochrobactrum spp., no similar
amplification products were detected in any microorganisms related genetically or antigenically to Brucella
spp. (Table 1). No cross-reactions were found using a
wide panel of microorganisms. The panel included
intracellular bacteria, some of which show a clear
tendency to cause bacteraemia, as well as others that
The panel of microorganisms against which our PCR
assay was tested is the widest studied to date and
included a strain of Bartonella spp. related phylogenetically to Brucella spp. that has not yet been studied.
These data reflect the high specificity of this PCR assay
and to some extent explain the low rate of false-positive results found in clinical studies in which it has been
tried [8, 9].
The existence of a cross-reaction with Ochrobactrum
spp. in our PCR assay is not surprising if we consider
that Ochrobactrum spp. is the closest known relative of
130
Table 2 PCR results with DNA from other non-Brucella organisms
Organisms (no. of isolates)
Source
PCR result
Acinetobacter baumannii (2)
Alcaligenes denitrificans (1)
Aeromonas hydrophila (1)
Aeromonas sobria (1)
Bacillus cereus (1)
Bacillus megaterium (1)
Bacillus subtilis (1)
Bacteroides fragilis (2)
Bordetella bronchiseptica (1)
Campylobacter spp. (2)
Candida albicans (2)
Candida glabrata (2)
Candida guillermondii (1)
Candida humicola (1)
Citrobacter freundii (1)
Corynebacterium spp. (1)
Enterobacter aerogenes (1)
Enterobacter cloacae (1)
Enterococcus faecalis (2)
Enterococcus faecium (1)
Escherichia coli (4)
Haemophilus influenzae (2)
Klebsiella pneumoniae (2)
Listeria monocytogenes (1)
Morganella morganii (1)
Mycobacterium tuberculosis (5)
Neisseria meningitidis (2)
Proteus mirabilis (2)
Providencia stuartii (1)
Pseudomonas aeruginosa (2)
Pseudomonas cepacia (1)
Salmonella spp. (2)
Serratia marcescens (2)
Shigella dysenteriae (1)
Staphylococcus aureus (2)
Staphylococcus epidermidis (2)
Staphylococcus haemolyticus (2)
Stenotrophomonas maltophilia (1)
Streptococcus agalactiae (2)
Streptococcus pneumoniae (2)
Streptococcus pyogenes (1)
HCH
CECT
CECT
HCH
CECT
CECT
CECT
HCH
CECT
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
CECT
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
HCH
CECT
HCH
HCH
HCM
HCH
HCH
HCH
HCH
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
CECT, Colección Española de Cultivos Tipo, Valencia, Spain;
HCH, Hospital Carlos Haya, Málaga, Spain
brucellae. In fact, Velasco et al. [13] assessed this
relatedness by whole-cell protein profiling, immunological methods and 16S rRNA sequence analysis. Da
Costa et al. [11] and Romero et al. [12] have reported
similar results using PCR assays with different DNA
targets specific to Brucella genus.
Three of the four strains of Ochrobactrum tested gave
positive results: LMG 3301, now redenominated
Ochrobactrum intermedium, LMG 3331 and CECT
4426. However, the DNA from a clinical strain of
Ochrobactrum spp. from a specimen collected in our
hospital gave a negative result. This strain was finally
identified as Ochrobactrum anthropi and was from a
patient with end-stage renal disease. The patient had a
peritoneal infection and was undergoing ambulatory
peritoneal dialysis. The reason for this negative result is
not completely clear, though it could be related to the
presumed heterogeneity existing in strains of Ochrobactrum anthropi. Recent studies have shown greater
genetic similarity between Brucella spp. and Ochrobactrum intermedium than between Brucella spp. and
Ochrobactrum anthropi. This observation would
explain why Ochrobactrum intermedium always gave
positive PCR results in our study, and in others,
whereas amplification of the strains of Ochrobactrum
anthropi has given variable results [11, 12].
We wondered how this cross-reaction might affect the
molecular diagnosis of brucellosis by means of PCR.
Ochrobactrum spp., formerly in CDC group Vd,
comprises a group of ubiquitous microorganisms that
appear to be distributed worldwide [14]. Although its
ecology is not well known, it has been isolated from
soil, water, multiple hospital materials and different
clinical specimens, and it may be part of the normal
flora of the large intestine. Although this microorganism would seem to occupy a microbial niche similar
to that of Pseudomonas aeruginosa, its pathogenic role
remains poorly understood. Since Ochrobactrum infection was first reported in the form of a pancreatic
abscess in 1980 [15], only 40 cases have been described
in the literature. Almost all cases occurred in severely
immunosuppressed patients or those with debilitating
illnesses. The infections were nosocomial, occurring in
patients with catheters or other foreign bodies [16, 17].
Thus, from a clinical point of view, this scenario is very
different compared with infection with Brucella spp.
With the exception of infections that occur in laboratory personnel, Brucella infection is always communityacquired and generally affects immunocompetent
subjects.
Since 1997, when we first started to work with this PCR
assay, none of the 9,735 clinically relevant cases of
bacteraemia detected in our hospital had been caused
by Ochrobactrum spp. Only 3 (0.0001%) of 24,471
isolates from other nonblood clinical samples were
identified as Ochrobactrum spp.; all three were from
two patients undergoing peritoneal dialysis. For these
reasons, we agree with others [12] that cross-reaction
between Brucella spp. and Onchrobactrum spp. in PCR
is unlikely and is of little clinical relevance.
Acknowledgements This research was supported by funds from
the Comisión Interministerial de Ciencia y Tecnología (CICYT)
Spain, and the European Commision (FEDER): Ref.
1FD97–0539. We also thank I. Johnstone for his help translating
the text.
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