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Applied and Environmental Microbiology, October 1998, p. 3748-3753, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Capacity of Nine Thermostable DNA Polymerases To Mediate DNA
Amplification in the Presence of PCR-Inhibiting Samples
Waleed
Abu Al-Soud and
Peter
Rådström*
Applied Microbiology, Center for Chemistry
and Chemical Engineering, Lund Institute of Technology, Lund
University, SE-221 00 Lund, Sweden
Received 20 March 1998/Accepted 6 July 1998
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ABSTRACT |
The PCR is an extremely powerful method for detecting
microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase from
Thermus aquaticus by many components found in
complex biological samples. In this study, we have compared the effects
of known PCR-inhibiting samples on nine thermostable DNA polymerases.
Samples of blood, cheese, feces, and meat, as well as various ions,
were added to PCR mixtures containing various thermostable DNA
polymerases. The nucleic acid amplification capacity of the nine
polymerases, under buffer conditions recommended by the manufacturers,
was evaluated by using a PCR-based detection method for Listeria
monocytogenes in the presence of purified template DNA and
different concentrations of PCR inhibitors. The AmpliTaq
Gold and the Taq DNA polymerases from Thermus
aquaticus were totally inhibited in the presence of
0.004% (vol/vol) blood in the PCR mixture, while the
HotTub, Pwo, rTth, and
Tfl DNA polymerases were able to amplify DNA in the
presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima
(Ultma) was found to be the most susceptible to PCR
inhibitors present in cheese, feces, and meat samples. When the
inhibitory effect of K and Na ions was tested on the nine polymerases,
HotTub from Thermus flavus and rTth
from Thermus thermophilus were the most resistant. Thus,
the PCR-inhibiting effect of various components in biological samples
can, to some extent, be eliminated by the use of the appropriate
thermostable DNA polymerase.
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INTRODUCTION |
The usefulness of PCR-based
detection of microorganisms in complex biological samples, such as
clinical, environmental, and food samples, is limited in part by the
presence of substances that inhibit the PCR or reduce the amplification
efficiency. The PCR inhibitors may act through one or more of the
following mechanisms (34): (i) interference with the cell
lysis step, (ii) degradation or capture of the nucleic acids, or (iii)
inactivation of the thermostable DNA polymerase.
A number of components have been reported to be PCR inhibitors, namely,
bile salts and complex polysaccharides in feces (17, 22),
heme in blood (2), humic substances in soil (30), proteinases in milk (25), and urea in urine (11).
Much effort is being devoted to the development of sample preparation
methods to overcome the problem of PCR inhibitors (for reviews, see
references 13 and 14). Different techniques are
being employed to reduce the effect of PCR inhibitors and/or to
separate the microorganisms from the PCR inhibitors. For example,
aqueous two phase-systems (15), boiling (23),
density gradient centrifugation (18), dilution
(5), DNA extraction methods (12), enrichment
media (31), filtration (6), and immunological
techniques (8) have been used to facilitate PCR. The
thermostable DNA polymerase is perhaps the most important target site
of PCR-inhibiting substances. The most widely used polymerase in
PCR-based methods for the detection of microorganisms is
Taq DNA polymerase from Thermus
aquaticus. The polymerase can be degraded by proteinases
(25), denatured by phenol (10) or detergents
(27), and inhibited by blocking of the active site by the
inhibitor, which is the effect of heme (2).
In a study by Wiedbrauk et al. (32), it was noted that both
Tfl and Tth DNA polymerases were more resistant
to aqueous and vitreous fluids of the eye than the polymerases
Taq, Tli, and Stoffel fragment. The aim of this
study was to systematically investigate the capacity of nine
commercially available thermostable DNA polymerases to amplify DNA in
the presence of certain complex biological samples and in the presence
of certain ions. The samples are all known to reduce the amplification
capacity of Taq DNA polymerase (13). We used a
PCR-based detection assay for Listeria monocytogenes to
study the effect of increasing the concentration of purified template
DNA and of adding different concentrations of the inhibitory samples.
Increasing the concentration of target DNA may be useful in overcoming
the effect of inhibitors (interfering with DNA and/or binding
reversibly to the DNA-binding domain of the polymerase), while adding
various concentrations of the inhibitory sample may be useful in
evaluating the effect of the inhibitory samples on the amplification
capacity of the nine polymerases.
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MATERIALS AND METHODS |
Bacterial strain and DNA extraction.
DNA of L. monocytogenes 167 vet (which was obtained from the Meat Research
Institute, Kävlinge, Sweden) was used as the target DNA in this
study. Extraction of L. monocytogenes DNA was performed in accordance with a standard technique described by Sambrook et al.
(28), modified by the addition of 30 U of mutanolysin (Sigma
Chemical Co., St. Louis, Mo.) per ml to the lysis solution. The
concentration of DNA was measured spectrophotometrically
(28), and the size of the L. monocytogenes
genomic DNA is estimated to be 3,150 kb (21).
PCR assay and incubation conditions.
The PCR assay was
carried out as previously described by Lantz et al. (15).
Briefly, the PCR assay was performed with the primer ru8
[5'-AAGGAGGTGATCCA(G/A)CCGCA(G/C)(G/C)TTC-3'], which is
complementary to the universal region of the 16S rRNA gene (26), and the primer LM2
(5'-CCTTTGACCACTCTGGAGACAGAGC-3'), which is complementary to
the L. monocytogenes-specific region of the 16S rRNA
gene (15). The 553-bp PCR product was visualized by 1.3%
agarose gel electrophoresis containing ethidium bromide (28). The gel was analyzed by using a gel documentation
system (Bio-Rad Laboratories, Hercules, Calif.). The results were
scored as follows: +, strong intensity of PCR product on agarose gel; ±, weak intensity of PCR product on agarose gel; 
, no visible PCR
product on agarose gel. Each of the PCR results given here was obtained
from at least two independent PCR amplifications.
The volume of the PCR mixture was 25 µl. All of the PCR mixtures
contained a 0.5 mM concentration of each of the primers ru8 and LM2 and
a 0.2 mM concentration of each of the deoxyribonucleoside triphosphates. Reaction buffers for the polymerases, as specified by
the manufacturers, were as follows: the PCR buffer of
AmpliTaq Gold DNA polymerase (Thermus flavus)
contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2,
and 0.75 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer
Cetus, Norwalk, Conn.); the PCR buffer of the Expand High Fidelity
(Expand HF) PCR system (which is a mixture of two DNA polymerases,
Taq [Thermus aquaticus] and
Pwo [Pyrococcus woesei]) contained 1× Expand
HF buffer, 1.5 mM MgCl2, and 1.33 U of Expand DNA
polymerase (Boehringer Mannheim GmbH, Mannheim, Germany); the PCR
buffer of HotTub DNA polymerase (Thermus ubiquatous) contained 50 mM Tris-HCl (pH 9.0), 20 mM
(NH4)2SO4, 2.5 mM
MgCl2, and 0.75 U of HotTub DNA polymerase
(Amersham Life Science, Cleveland, Ohio); the PCR buffer of
Pwo DNA polymerase (Pyrococcus woesei) contained
10 mM Tris-HCl (pH 8.85; 20°C), 25 mM KCl, 5 mM
(NH4)2SO4, 1.5 mM
MgCl2, and 1.25 U of Pwo DNA polymerase
(Boehringer Mannheim); the PCR buffer of rTth DNA polymerase (Thermus thermophilus) contained 5% (vol/vol) glycerol, 10 mM Tris-HCl (pH 8.3), 0.1 M KCl, 0.05% (wt/vol) Tween 20, 0.75 mM EGTA, 2.5 mM MgCl2, and 1.25 U of rTth DNA
polymerase (Perkin-Elmer Cetus); the PCR buffer of Taq DNA
polymerase (Thermus aquaticus) contained 10 mM
Tris-HCl, 1.5 mM MgCl2, 50 mM KCl (pH 8.3; 20°C), and
0.75 U of Taq DNA polymerase (Boehringer Mannheim); the PCR buffer of Tfl DNA polymerase (Thermus flavus)
contained 20 mM Tris-Acetate (pH 9.0), 10 mM
(NH4)2SO4, 75 mM potassium acetate, 0.05% Tween 20, 2.5 mM MgSO4, and 0.5 U of Tfl
DNA polymerase (Promega Corporation, Madison, Wis.); the PCR buffer of
Tli DNA polymerase (Thermus litoralis) contained
10 mM Tris-HCl (pH 9.0; 25°C), 0.1% Triton X-100, 50 mM KCl, 2.5 mM
MgCl2, and 0.3 U of Tli DNA polymerase
(Promega); the PCR buffer of Ultma DNA polymerase (Thermotoga maritima) contained 10 mM Tris-HCl (pH 8.8; room
temperature), 10 mM KCl, 0.002% (vol/vol) Tween 20, 2.5 mM
MgCl2, and 0.75 U of Ultma DNA polymerase
(Perkin-Elmer Cetus). The reaction mixtures were subjected to 30 amplification cycles consisting of heat denaturation at 94°C for
40 s, primer annealing at 53°C for 40 s, and DNA extension at 72°C for 40 s. Finally, the mixtures were maintained at
72°C for 7 min for the final extension of DNA. These incubation
conditions were the same for all amplification reaction mixtures,
except those containing AmpliTaq Gold, since this polymerase
requires a hot start (95°C for 10 min). Incubation was carried out in
a model 2400 thermal cycler (Perkin-Elmer Cetus).
PCR-inhibitory samples.
The blood sample used was drawn from
a healthy person into 10-ml evacuated blood-collecting tubes containing
0.1 ml of 0.47 M EDTA (Terumo Europe N. V., Leuven, Belgium). The
Danish Blue Castello soft cheese (henceforth referred to as cheese) was
diluted 10-fold in physiological saline solution and homogenized for 2 min in a stomacher (Lab-Blender 400; Steward Laboratory, London, United
Kingdom). The fecal sample was obtained from a healthy person, diluted
10-fold in physiological saline solution, and homogenized for 2 min in
the stomacher. The minced pork meat (henceforth referred to as meat)
was diluted 10-fold in physiological saline solution and homogenized
for 2 min in the stomacher. Each of the PCR-inhibitory samples was
poured into a sterile 1.5-ml Eppendorf tube and stored at 20°C. The
frozen blood and cheese, feces, and meat homogenates were thawed at
room temperature, mixed with a vortex mixer, and allowed to stand for 5 min to allow large particles to settle before they were added directly
or after dilution to the reaction mixtures of the nine polymerases.
The effect of target DNA concentration on sensitivity in the presence
of PCR-inhibitory samples was studied by adding different
concentrations of
L. monocytogenes DNA (1 µg to 10 fg
per reaction
tube) to reaction mixtures containing 20% (vol/vol)
blood, 20%
(vol/vol) cheese homogenate, 20 or 0.4% (vol/vol) fecal
homogenate,
and 20 or 0.2% (vol/vol) meat homogenate.
To study the effect of different concentrations of the PCR-inhibitory
samples on the amplification capacity of the nine polymerases,
different blood concentrations (20, 2, 0.2, 0.04, 0.02, 0.004,
and
0.002%) and different cheese, feces, and meat homogenate
concentrations
(20, 2, 0.2, 0.1, 0.07, 0.05, and 0.04%) were added to
PCR mixtures
of the nine polymerases containing 1 ng of
L. monocytogenes DNA
per reaction tube.
Ion solutions.
Different concentrations of
CaCl2 · 2H2O, KCl,
MgCl2 · 6H2O, and NaCl (Merck,
Darmstadt, Germany) were prepared, as sterile solutions, to study their
effect on the amplification capacity of the nine polymerases. The ion
concentrations were 1, 5, 10, 20, 30, 40, and 50 mM CaCl2;
10, 50, 100, 200, 300, 400, 500, and 750 mM KCl; 25, 50, 75, 100, and
150 mM MgCl2; and 50, 100, 200, 300, 400, 500, and 750 mM
NaCl. Five microliters of the different ion concentrations were added
to the PCR mixtures of the nine polymerases containing 1 ng of
L. monocytogenes DNA per reaction tube.
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RESULTS AND DISCUSSION |
Amplification capacity of the nine thermostable DNA
polymerases.
The DNA amplification capacities of nine thermostable
DNA polymerases were compared in accordance with a PCR-based assay
previously designed to detect L. monocytogenes
(15). To assess the detection limit for L. monocytogenes DNA in PCR-inhibitory samples relative to the target
DNA in the absence of the sample (i.e., in pure water), various amounts
of DNA were added to the PCR mixtures containing different polymerases
(Table 1). Six of the nine polymerases detected 0.1 ng of DNA. The detection limits for the polymerases Expand, Tli, and Ultma were 1 pg, 10 pg, and 1 ng
per reaction tube, respectively.
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TABLE 1.
Inhibitory effects of blood, cheese, feces, and meat on
the amplification capacities of nine thermostable DNA polymerases
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When blood was added to the PCR mixtures, it was found to be
highly inhibitory to the DNA polymerases from
Thermus
aquaticus (Ampli
Taq Gold and
Taq)
and
Thermotoga maritima (
Ultma) (Table
1). The
addition of 20% (vol/vol) blood was completely inhibitory
to these
polymerases, even after the concentration of
L. monocytogenes DNA was increased to 1 µg per reaction tube.
However, the polymerases
Expand, Hot
Tub,
Pwo,
rTth,
Tfl, and
Tli successfully
amplified
the selected region of the 16S rRNA gene in the presence of
20%
(vol/vol) blood. The amplification sensitivities of four of these
six polymerases were not affected by the presence or absence of
blood
in the PCR, while the sensitivity of Expand was decreased
by 5 log
units and the sensitivity of
Tli was decreased by 2 log
units in the presence of 20% (vol/vol) blood. Akane et al.
(
2)
found that the heme compound of blood is an inhibitor
for Ampli
Taq DNA polymerase, and they showed that this
inhibition was not due
to irreversible inactivation of the polymerase.
Heme has been
suggested to form a stable complex with the DNA
polymerase, thereby
preventing formation, and also causing
dissociation, of the DNA
polymerase complex (
4).
The addition of 20% (vol/vol) cheese homogenate decreased the
sensitivity of the polymerases Ampli
Taq Gold, Expand, and
Pwo by 1 log unit and the sensitivity of the polymerase
Ultma by 2
log units, while the detection sensitivities of
the other polymerases
in the presence of 20% (vol/vol) cheese
homogenate were the same
as for pure water solutions of DNA (Table
1).
When 20% (vol/vol)
fecal homogenate or 20% (vol/vol) meat homogenate
was added to
reaction mixtures of the nine polymerases containing
different
concentrations of
L. monocytogenes DNA (1 µg to 0.1 pg per reaction
tube), the nine polymerases were completely
inhibited (data not
shown). Decreasing the fecal concentration to 0.4%
(vol/vol) resulted
in a marked difference between the polymerases
(Table
1), so
that 0.4% (vol/vol) fecal homogenate was found not to be
inhibitory
to the polymerases
Pwo, from
Pyrococcus
woesei, and
rTth, from
Thermus thermophilus,
and they exhibited the same sensitivity
as for pure water solutions of
DNA. On the other hand, the other
polymerases had a sensitivity to 1 µg of
L. monocytogenes DNA
per reaction tube in the
presence of 0.4% (vol/vol) fecal homogenate.
Decreasing the meat
homogenate concentration to 0.2% (vol/vol)
gave rise to a small
difference between the sensitivities of the
polymerases (Table
1). The
polymerase
Tfl, from
Thermus flavus,
was slightly
inhibited by the addition of 0.2% (vol/vol) meat
homogenate, such that
it had a sensitivity 1 log unit less than
pure water solutions of DNA,
while the polymerase
Ultma required
an increase in the
concentration of
L. monocytogenes DNA to 0.1
µg per
reaction tube to overcome the inhibitory effect of 0.2%
(vol/vol) meat
homogenate. The rest of the polymerases exhibited
a sensitivity to 10 ng of
L. monocytogenes DNA per reaction. These
results
agree with previous studies in which both fecal and meat
homogenates
were found to be highly inhibitory to Ampli
Taq and
Taq DNA polymerases (
17,
19,
33,
35).
Effect of different concentrations of blood, cheese, feces,
and meat on the DNA polymerases.
The polymerases
HotTub, Tfl, and Tli amplified the
selected region of L. monocytogenes DNA in the presence
of all tested concentrations of blood (Table
2). Although the polymerases
Pwo and rTth were not inhibited in the presence
of 20% (vol/vol) blood, and the polymerases Expand and
Ultma were not inhibited in the presence of 2% (vol/vol)
blood, a PCR-inhibitory span was observed for the polymerases
Pwo (0.02%), rTth (2 to 0.02%), and
Ultma (0.2 to 0.02%). A similar range of PCR inhibition was
noted when we studied the effect of adding different concentrations of
culture medium containing 28.6% whole blood to PCR mixtures containing Taq DNA polymerase and Streptococcus pneumoniae
DNA (1). The addition of 0.5 µg of bovine serum albumin
counteracted the inhibitory effect of the blood culture at various
concentrations. A possible explanation of this may be the ability of
bovine serum albumin to bind heme and/or serve as a competitive
target for proteinases. On the other hand, the concentration of blood
had to be reduced to as little as 0.002% (vol/vol) for the
polymerases AmpliTaq Gold and Taq to regain their
amplification capacity in the presence of 1 ng of L. monocytogenes DNA (Table 2). In a study by Mercier et al.
(20), it was found that the addition of 4% (vol/vol) blood
to the reaction mixture was totally inhibitory to Taq DNA polymerase, whereas PCR amplification took place in the presence of
1 or 2% (vol/vol) blood. Panaccio and Lew (24),
however, found that the addition of 1% (vol/vol) blood was totally
inhibitory to Taq DNA polymerase, while they succeeded in
amplifying a target sequence in the presence of up to 4%
(vol/vol) blood by using Tth DNA polymerase. A possible
explanation of these conflicting results for the effect of blood on
Taq DNA polymerase may be the use of different target DNA
concentrations and PCR conditions.
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TABLE 2.
Inhibitory effects of different blood concentrations on
the amplification capacities of nine thermostable DNA polymerases
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When different concentrations of the cheese homogenate were added to
PCR mixtures of the nine polymerases containing 1 ng
of
L. monocytogenes DNA (Table
3), eight
of the nine polymerases
were not inhibited by the cheese homogenate,
whereas the amplification
capacity of the polymerase
Ultma
was totally inhibited at concentrations
of cheese homogenate over 0.2%
(vol/vol). The generally low level
of inhibition by cheese may be due
to the choice of the particular
cheese used. In a previous study,
variations in PCR inhibition
were observed for different soft cheeses
(
16).
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TABLE 3.
Inhibitory effects of different concentrations of cheese,
feces, and meat on the amplification capacities of nine
thermostable DNA polymerases
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The concentration of fecal homogenate had to be reduced to 0.2%
(vol/vol) to detect the PCR product in both replicates for
the
polymerases Ampli
Taq Gold, Expand,
Pwo, and
rTth (Table
3).
The amplification capacity of the
polymerases Hot
Tub,
Taq,
Tfl,
and
Ultma was restored after the fecal homogenate concentration
was reduced to 0.1% (vol/vol). The polymerase most sensitive to
fecal
homogenate was
Tli, which required a decrease in the
concentration
of fecal homogenate to 0.07% (vol/vol) to restore its
amplification
capacity. In the case of the meat homogenate, a
concentration
as low as 0.04% (vol/vol) was inhibitory to the
polymerase
Ultma (Table
3). On the other hand, the
amplification capacities of
the polymerases Expand,
rTth,
and
Tfl were restored when the concentration
of meat
homogenate was decreased to 0.2% (vol/vol). The PCR product
was
detected with the remaining polymerases in tubes containing
meat
homogenate at concentrations of, but not higher than, 0.1%
(vol/vol).
Effect of trypsin inhibitor on rTth inhibitors in
blood.
To investigate whether a trypsin inhibitor relieves
inhibition of rTth DNA polymerase in PCR mixtures with 2 to
0.02% (vol/vol) blood concentrations (see Table 2), 5 µg of lima
bean trypsin inhibitor (Sigma Chemical Co.) was added to PCR
mixtures containing different concentrations of blood and 1 ng of
L. monocytogenes DNA. The addition of trypsin inhibitor
decreased the range of inhibitory blood concentrations relative to
those shown in Table 2. However, blood concentrations of 0.2 and 0.04%
(vol/vol) were still inhibitory to the rTth DNA
polymerase (Fig. 1). Powell et al.
(25) have also shown that the addition of proteinase
inhibitors to the reaction mixtures can partially counteract
the effect of PCR inhibitors in milk. They studied the detection of
L. monocytogenes DNA in PCR mixtures with various
concentrations of milk and found the PCR to be inhibited by the
milk in the concentration range of 0.5 to 0.01% (vol/vol). They
suggested that a proteinase which inhibits the PCR by degrading the DNA
polymerase may be present in milk.
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FIG. 1.
Relief of rTth inhibition due to blood by the
addition of 5 µg of lima bean trypsin inhibitor in a PCR
mixture containing 1 ng of L. monocytogenes DNA
and blood at different concentrations. Lanes: 1, 100-bp molecular
weight marker (Boehringer Mannheim); 2, negative control; 3, positive
control; 4, 20% blood; 5, 2% blood; 6, 0.2% blood; 7, 0.04% blood;
8, 0.02% blood; 9, 0.004% blood.
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Effect of ions on the nine thermostable DNA polymerases.
Different concentrations of ions were added to reaction mixtures
containing 1 ng of L. monocytogenes DNA to study their
effect on the amplification capacity of the nine polymerases (Tables 4
to 7). The inhibitory effect of divalent
ions (Ca and Mg) was more pronounced than that of monovalent ions (K
and Na), with Ca being the most inhibitory. The polymerase most
sensitive to K, Mg, and Na was Ultma from Thermatoga
maritima, whereas the polymerases AmpliTaq Gold and
Taq were the most sensitive to Ca. On the other hand, the
polymerases from Thermus flavus (HotTub) and
Thermus thermophilus (rTth) were the most
resistant to the monovalent ions, while the polymerase
HotTub was the most resistant to Ca, which can be related to
the low ionic content of HotTub buffer. The levels of
resistance of the polymerase Taq to K, Mg, and Na were
higher than that of the polymerase AmpliTaq Gold, although
they are derived from the same organism. The resistance pattern of the
polymerase Expand, which is a mixture of two polymerases (Pwo and Taq), to K, Mg, and Na was more similar
to that of Taq than to that of Pwo. However, the
polymerase Expand was more resistant to Ca ions than were the
polymerases Taq and Pwo. It has previously been
found that the addition of K ions at concentrations higher than 75 mM
was completely inhibitory to amplification of DNA by AmpliTaq (9), and increasing the concentration of
Mg ions to 15 mM has been found to inhibit the ability of
Taq DNA polymerase to detect herpes simplex virus
(32). The highly inhibitory effect of the Ca ion may be due
to its ability to compete with the Mg ion. Ca ion concentrations above
3 mM have previously been found to be inhibitory to the amplification
capacity of AmpliTaq DNA polymerase. This inhibitory effect
was reversed by increasing the Mg ion concentration in the reaction
mixture (3). Also, it has previously been shown that
variations in the performance of DNA polymerases in coamplification PCR
were salt dependent (7).
Conclusions.
The inhibition of Taq DNA polymerase
amplification by the components of biological samples can be reduced or
eliminated by the use of an appropriate thermostable DNA polymerase. A
number of DNA polymerases are now commercially available. These
polymerases exhibit different properties with regard to resistance to
various components in clinical, environmental, and food samples. By
selecting an appropriate polymerase, it is possible to more efficiently amplify nucleic acid in the presence of biological material without the
need for extensive sample processing prior to PCR.
Based on the results given in Tables
1 and
2, eight of the nine
polymerases can be divided into three groups: (i) the
blood-sensitive
polymerases (Ampli
Taq Gold,
Taq, and
Ultma), i.e., those whose
amplification capacities were not restorable by either increasing
the
template DNA concentration (Table
1) nor decreasing the blood
concentration (Table
2); (ii) the blood-resistant polymerases
(Hot
Tub,
Tfl, and
Tli), which
amplified the template DNA in the
presence of all tested blood
concentrations; and (iii) the feces-resistant
polymerases
(
Pwo and
rTth), which can amplify DNA in the
presence
of 20% blood but have a range of inhibition around the blood
concentration
of 0.02%. The polymerase Expand, which is a mixture of
Pwo and
Taq, has a resistance pattern to the
inhibitory samples that is
different from the other eight enzymes.
We have shown that components in biological samples can differentially
inhibit different polymerases in the PCR, oftentimes
by several orders
of magnitude. However, many intrinsic factors
may also play a role in
the resistance of the thermostable DNA
polymerases to PCR inhibition,
for instance, the difference in
enzyme purification techniques, whether
the polymerase is native
or recombinant, and the buffer content.
Taq DNA polymerase from
different commercial sources has
previously been reported to be
inhibited differentially by humic
substances in soil extracts
(
29). Although not examined in
this study, the DNA extraction
protocol and the presence of trace
levels of extraction reagents
in the purified DNA can also affect
extraction efficiency and
increase PCR inhibition, thereby
affecting the sensitivity of
detection. The polymerase
Tth
has been found by Katcher and Schwartz
(
10) to maintain both
DNA- and RNA-dependent DNA polymerase
activities in the presence of 5%
(vol/vol) phenol, while a trace
amount of phenol was found to be
inhibitory to
Taq DNA polymerase.
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ACKNOWLEDGMENTS |
We thank the following companies for donating the DNA polymerases
used in this study: Amersham Life Science, Boehringer Mannheim, Perkin-Elmer Cetus, and Promega.
This work was supported by a grant from the Swedish Council for
Forestry and Agricultural Research.
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FOOTNOTES |
*
Corresponding author. Mailing address: Applied
Microbiology, Center for Chemistry and Chemical Engineering, Lund
Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: 46 46 222 34 12. Fax: 46 46 222 42 03. E-mail: Peter.Radstrom{at}tmb.lth.se.
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REFERENCES |
| 1.
|
Abu Al-Soud, W.,
P.-G. Lantz,
A. Bäckman,
P. Olcén, and P. Rådström.
1998.
A sample preparation which facilitates detection of bacteria in blood cultures by the polymerase chain reaction.
J. Microbiol. Methods
32:217-224.
|
| 2.
|
Akane, A.,
K. Matsubara,
H. Nakamura,
S. Takahashi, and K. Kimura.
1994.
Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from blood strains, a major inhibitor of polymerase chain reaction (PCR) amplification.
J. Forensic Sci.
39:362-372[Medline].
|
| 3.
|
Bickley, J.,
J. K. Short,
D. G. McDowell, and H. C. Parkes.
1996.
Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions.
Lett. Appl. Microbiol.
22:153-158[Medline].
|
| 4.
|
Byrnes, J. J.,
K. M. Downey,
L. Esserman, and A. G. So.
1975.
Mechanism of hemin inhibition of erythroid cytoplasmic DNA polymerase.
Biochemistry
14:796-799[Medline].
|
| 5.
|
Chernesky, M. A.,
D. Jang,
L. Sellors,
K. Luinstra,
S. Chong,
S. Castriciano, and J. B. Mahony.
1997.
Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women.
Mol. Cell Probes
11:243-249[Medline].
|
| 6.
|
DiMichele, L. J., and M. J. Lewis.
1993.
Rapid, species-specific detection of lactic acid bacteria from beer using the polymerase chain reaction.
J. Am. Brew. Chem. Soc.
51:63-66.
|
| 7.
|
Favre, N., and W. Rudin.
1996.
Salt-dependent performance variation of DNA polymerases in co-amplification PCR.
BioTechniques
21:28-30[Medline].
|
| 8.
|
Fluit, A. C.,
R. Terensma,
M. J. C. Visser,
C. J. M. Aarsman,
M. J. J. G. Poppelier,
B. H. I. Keller,
P. Klapwijk, and J. Verhoef.
1993.
Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.
Appl. Environ. Microbiol.
59:1289-1293[Abstract/Free Full Text].
|
| 9.
|
Innis, M. A.,
K. B. Myambo,
D. H. Gelfand, and M. A. D. Brow.
1988.
DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.
Proc. Natl. Acad. Sci. USA
85:9436-9440[Abstract/Free Full Text].
|
| 10.
|
Katcher, H. L., and I. Schwartz.
1994.
A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol.
BioTechniques
16:84-92[Medline].
|
| 11.
|
Khan, G.,
H. O. Kangro,
P. J. Coates, and R. B. Heath.
1991.
Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA.
J. Clin. Pathol.
44:360-365[Abstract/Free Full Text].
|
| 12.
|
Klein, A.,
R. Barsuk,
S. Dagan,
O. Nusbaum,
D. Shouval, and E. Galun.
1997.
Comparison of methods for extraction of nucleic acids from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences.
J. Clin. Microbiol.
35:1897-1899[Abstract].
|
| 13.
|
Lantz, P.-G.
1998.
Ph.D. thesis.
Lund University, Lund, Sweden.
|
| 14.
|
Lantz, P.-G.,
B. Hahn-Hägerdal, and P. Rådström.
1994.
Sample preparation methods in PCR-based detection of food pathogens.
Trends Food Sci. Technol.
5:384-389.
|
| 15.
|
Lantz, P.-G.,
F. Tjerneld,
E. Borch,
B. Hahn-Hägerdal, and P. Rådström.
1994.
Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method.
Appl. Environ. Microbiol.
60:3416-3418[Abstract/Free Full Text].
|
| 16.
|
Lantz, P.-G.,
F. Tjerneld,
B. Hahn-Hägerdal, and P. Rådström.
1996.
Use of aqueous two-phase system in sample preparation for polymerase chain reaction-based detection of microorganisms.
J. Chromatogr. B
680:165-170[Medline].
|
| 17.
|
Lantz, P.-G.,
M. Matsson,
T. Wadström, and P. Rådström.
1997.
Removal of PCR inhibitors from human fecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR.
J. Microbiol. Methods
28:159-167.
|
| 18.
|
Lindqvist, R.,
B. Norling, and S. T. Lambertz.
1997.
A rapid sample preparation method for detection of food pathogens based on buoyant density centrifugation.
Appl. Microbiol.
24:306-310.
|
| 19.
|
Marsiglia, M. L.,
N. Ikuta,
A. K. Fonseca,
D. T. Schuch,
I. Hötzel,
L. S. Ozaki,
E. K. Marques, and V. R. Lunge.
1997.
Development of a combined selective enrichment method and polymerase chain reaction (PCR) assay for sensitive detection of Salmonella in food samples.
J. Microbiol. Biotechnol.
13:649-654.
|
| 20.
|
Mercier, B.,
C. Gaucher,
O. Feugeas, and C. Mazurier.
1990.
Direct PCR from whole blood, without DNA extraction.
Nucleic Acids Res.
18:5908[Free Full Text].
|
| 21.
|
Michel, E., and P. Cossart.
1992.
Physical map of the Listeria monocytogenes chromosome.
J. Bacteriol.
174:7098-7103[Abstract/Free Full Text].
|
| 22.
|
Monteiro, L.,
D. Bonnemaison,
A. Vekris,
K. G. Petry,
J. Bonnet,
R. Vidal,
J. Cabrita, and F. A. Megraud.
1997.
Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.
J. Clin. Microbiol.
35:995-998[Abstract].
|
| 23.
|
Olcén, P.,
P.-G. Lantz,
A. Bäckman, and P. Rådström.
1995.
Rapid diagnosis of bacterial meningitis by a seminested PCR strategy.
Scand. J. Infect. Dis.
27:537-539[Medline].
|
| 24.
|
Panaccio, M., and A. Lew.
1991.
PCR based diagnosis in the presence of 8% (v/v) blood.
Nucleic Acids Res.
19:1151[Free Full Text].
|
| 25.
|
Powell, H. A.,
C. M. Gooding,
S. D. Garrett,
B. M. Lund, and R. A. McKee.
1994.
Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction.
Lett. Appl. Microbiol.
18:59-61.
|
| 26.
|
Rådström, P.,
A. Bäckman,
N. Qian,
P. Kragsbjerg,
C. Påhlson, and P. Olcén.
1994.
Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy.
J. Clin. Microbiol.
32:2738-2744[Abstract/Free Full Text].
|
| 27.
|
Rossen, L.,
P. Nørskov,
K. Holmstrømet, and O. F. Rasmussen.
1992.
Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions.
Int. J. Food Microbiol.
17:37-45[Medline].
|
| 28.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 29.
|
Tebbe, C., and W. Vahjen.
1993.
Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.
Appl. Environ. Microbiol.
59:2657-2665[Abstract/Free Full Text].
|
| 30.
|
Tsai, Y.-L., and B. H. Olson.
1992.
Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction.
Appl. Environ. Microbiol.
58:754-757[Abstract/Free Full Text].
|
| 31.
|
Wernars, K.,
E. Delfgou,
P. S. Soentoro, and S. Notermans.
1991.
Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction.
Appl. Environ. Microbiol.
57:1914-1919[Abstract/Free Full Text].
|
| 32.
|
Wiedbrauk, D. L.,
J. C. Werner, and A. M. Drevon.
1995.
Inhibition of PCR by aqueous and vitreous fluids.
J. Clin. Microbiol.
33:2643-2646[Abstract].
|
| 33.
|
Wilde, J.,
J. Eiden, and R. Yolken.
1990.
Removal of inhibitory substances from human fecal speciments for detection of group A retroviruses by reverse transcriptase and polymerase chain reactions.
J. Clin. Microbiol.
28:1300-1307[Abstract/Free Full Text].
|
| 34.
|
Wilson, I. G.
1997.
Inhibition and facilitation of nucleic acid amplification.
Appl. Environ. Microbiol.
63:3741-3751[Medline].
|
| 35.
|
Witham, P. K.,
C. T. Yamashiro,
K. J. Livak, and C. A. Batt.
1996.
A PCR-based assay for detection of Escherichia coli Shiga-like toxin genes in ground beef.
Appl. Environ. Microbiol.
62:1347-1353[Abstract].
|
Applied and Environmental Microbiology, October 1998, p. 3748-3753, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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-
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-
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Abstract
Introduction
