Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

PCR inhibitors – occurrence, properties and removal

C. Schrader1, A. Schielke2, L. Ellerbroek1 and R. Johne1
1 Food Hygiene and Safety Concepts, Federal Institute for Risk Assessment, Berlin, Germany
2 Department for Infectious Disease Epidemiology, Robert Koch Institute, Berlin, Germany

Keywords Summary
detection, environmental, food, polymerase
chain reaction. The polymerase chain reaction (PCR) is increasingly used as the standard
method for detection and characterization of microorganisms and genetic
Correspondence markers in a variety of sample types. However, the method is prone to
Reimar Johne, Federal Institute for Risk inhibiting substances, which may be present in the analysed sample and which
Assessment, Max-Dohrn-Strasse 8-10, 10589
may affect the sensitivity of the assay or even lead to false-negative results. The
Berlin, Germany. E-mail: reimar.johne@bfr.
PCR inhibitors represent a diverse group of substances with different
bund.de
properties and mechanisms of action. Some of them are predominantly found
2012/0617: received 5 April 2012, revised 14 in specific types of samples thus necessitating matrix-specific protocols for
June 2012 and accepted 27 June 2012 preparation of nucleic acids before PCR. A variety of protocols have been
developed to remove the PCR inhibitors. This review focuses on the general
doi:10.1111/j.1365-2672.2012.05384.x properties of PCR inhibitors and their occurrence in specific matrices.
Strategies for their removal from the sample and for quality control by
assessing their influence on the individual PCR test are presented and
discussed.

In the following sections, the different classes of PCR

Background
inhibitors and their mechanisms of action are presented.
In the last few decades, the polymerase chain reaction The incidence of PCR inhibitors in different types of
(PCR) has become one of the most powerful molecular matrices such as clinical, food or environmental speci-
biological tools. PCR may be used for diagnosis of infec- mens will be reviewed. Finally, different possibilities to
tious or hereditary diseases and for genetic analyses in a remove PCR inhibitors as well as strategies for quality
large variety of sample types. The PCR is a very rapid controls of the PCR will be presented and discussed.
and sensitive method, in which genomic DNA is expo-
nentially amplified by a DNA polymerase using specific
Substance classes of PCR inhibitors
primer molecules. Variations of the PCR comprise the
reverse transcription (RT-) PCR using RNA as template, PCR inhibitors are a very heterogeneous group of chemi-
which is first transcribed into DNA by a reverse trans- cal substances. One certain matrix may contain many dif-
criptase, or real-time PCR, which uses fluorescent probes ferent inhibitory substances and the same inhibitors can
for the detection of the PCR product providing quantita- be found in many different matrices. Organic as well as
tive information. The PCR is an enzymatic reaction and inorganic substances, which may be dissolved or solid,
therefore sensitive to inhibitors. The occurrence of such can appear as PCR inhibitors. Calcium ions are an exam-
so-called PCR inhibitors, which comprise all substances ple for inorganic substances with inhibitory effects on the
that have a negative effect on the PCR, is a major draw- PCR. However, most of the known inhibitors are organic
back of the PCR. PCR inhibitors can originate from the compounds, for example, bile salts, urea, phenol, ethanol,
sample or may be introduced during sample processing polysaccharides, sodium dodecyl sulphate (SDS), humic
or nucleic acid extraction. The major consequence of a acids, tannic acid, melanin as well as different proteins,
partly or total inhibition of the PCR is a decreased such as collagen, myoglobin, haemoglobin, lactoferrin,
sensitivity or false-negative results, respectively. immunoglobin G (IgG) and proteinases (Rossen et al.

© 2012 The Authors

1014 Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology
C. Schrader et al. PCR inhibitors

1992; Rådström et al. 2004). Besides the substance class, 2000). The DNA used as template of the PCR can be
the concentration of the compound is important for its modified or degraded by nucleases and other substances.
inhibitory effect. For a detailed review on the relation of Annealing of the primers to the DNA template may be
the concentration of inhibitors (e.g. sodium chloride, disturbed by certain PCR inhibitors (Chandler et al.
magnesium chloride, sucrose, phenol, SDS, ethanol or 1998; Abbaszadegan et al. 1999). As this effect is because
cetrimonium bromide) and their inhibitory effects, see of a competitive binding of the inhibitor to the template,
the article of Rossen et al. (1992). appropriate primer design leading to higher melting
PCR inhibitors can be found in a variety of biological points can overcome this problem (Huggett et al. 2008;
materials (organs, blood, body fluids etc.), environmental Opel et al. 2010).
samples (water, soil, air etc.) and food (meat, milk, fruits, There are many PCR inhibitors that target the DNA
vegetables, seafood etc.). In addition, inhibitory polymerase directly or indirectly. Proteases or detergents
substances may also be unintentionally added during present in the reaction can degrade this enzyme (Rossen
transport, sample processing (e.g. pre-concentration et al. 1992; Powell et al. 1994). For example, urea
procedures) or nucleic acid extraction. Table 1 shows (Saulnier and Andremont 1992; Wilson 1997) and phenol
some examples of matrices and their typical inhibitors. (Katcher and Schwartz 1994) are known to degrade DNA
polymerases. Calcium, collagen, haematin and tannic acid
may inhibit polymerase activity (Opel et al. 2010). Mela-
Mechanisms of action of PCR inhibitors
nin forms a reversible complex with the DNA polymerase
PCR inhibitors may interfere with different steps of a (Eckhart et al. 2000) and polysaccharides may disturb the
PCR analysis (Fig. 1). Generally, several PCR compo- enzymatic process by mimicking the structure of nucleic
nents, especially DNA, may adsorb to polymeric surfaces, acid (Peist et al. 2001). Humic acids interact with the
for example, to the wall of vessels and reaction tubes, template DNA and the polymerase thus preventing the
during sample processing, extraction or during PCR enzymatic reaction even at low concentrations (Sutlović
(Butot et al. 2007b; Fox et al. 2007; Gassilloud et al. et al. 2005, 2008). Other substances react with cofactors
2007; Gonzalez et al. 2007). The efficacy of sample pro- of the polymerase. High concentrations of calcium may
cessing and nucleic acid extraction may be affected. Nuc- lead to a competitive binding by the DNA polymerase
leases may degrade template RNA or DNA. Phenols may instead of magnesium and complexing agents, for exam-
cross-link RNA under oxidizing conditions and thus ple, tannic acid, deplete magnesium. In both cases, mag-
hamper RNA isolation (Su and Gibor 1988; Wilkins and nesium is no longer available as cofactor for the
Smart 1996). In addition, the existence of polysaccharides polymerase and its activity is decreased (Opel et al.
may reduce the capacity to resuspend precipitated RNA 2010). For real-time PCR assays, the interference with the
(Wilkins and Smart 1996; Sipahioglu et al. 2006). Reverse fluorescent probes or increased background fluorescence
transcription may be inhibited, for example, by direct represents additional mechanisms of action for PCR
interaction of the enzyme with melanin (Eckhart et al. inhibitors decreasing sensitivity. Examples of PCR
Table 1 Selected matrices and their identified polymerase chain reaction inhibitors

Matrix Contained inhibitors References

Clinical specimens Antiviral substances (e.g. acyclovir), Al-Soud and Rådström (2001); Burkardt (2000); Rådström et al. (2004);
(e.g. blood; Haemoglobin, Heparin, Hormones, Yedidag et al. (1996)
muscle tissues) IgG, Lactoferrin, Myoglobin
Stool Complex polysaccharides, Bile Kreader (1996); Monteiro et al. (1997); Rådström et al. (2004); Chaturvedi
salts, Lipids, Urate et al. (2008)
Seafood, bivalves, Algae, Glycogen, Polysaccharides Atmar et al. (1993, 1995); Richards (1999)
oysters
Berries Phenols, Polysaccharides Seeram et al. (2006); Wei et al. (2008)
Plants Pectin, Polyphenols, Polysaccharides, Demeke and Adams (1992); Henson and French (1993); John (1992);
Xylan Sipahioglu et al. (2006); Su and Gibor (1988); Wan and Wilkins (1994); Wei
et al. (2008); Wilkins and Smart 1996
Cheese, milk Proteases (e.g. plasmin), Calcium ions, Bickley et al. (1996); Powell et al. (1994); Rossen et al. (1992)
Water, environment Debris, Fulmic acids, Humic acids, Humic Abbaszadegan et al. (1993); Ijzerman et al. (1997)
material Metal ions, Polyphenol
Palaeobiology, Bone dust, Coprolite Peat Baar et al. (2011)
archaeology, extract, Clay-rich soil
forensic

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Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology 1015
PCR inhibitors C. Schrader et al.

The most critical component in urine samples is urea,

1 250
which may lead to the degradation of the polymerase
200

150
(Saulnier and Andremont 1992; Wilson 1997). However,
100

the effect is dependant on the concentration of urea in

the sample and initiates with a concentration of about
50 mmol l 1 (Khan et al. 1991). As the urine excretion
in humans is – dependent on diet and age – in the range
between 340 and 580 mmol urea per day, inhibitory
2 concentrations are not unusual.
Stool and faecal samples contain highly variable com-
ponents dependant on nutrition, gut flora, lifestyle and
environment of the patient (Oikarinen et al. 2009).
3
Inhibitors may include polysaccharides or chlorophyll
originating from herbs and vegetables, bile salts, urea,
glycolipids, haemoglobin and heparin (Lantz et al. 1997;
RT
Monteiro et al. 1997; Pontiroli et al. 2011).
The most important inhibitory factors present in bile
samples are the bile acids and their corresponding salts.
However, efficient amplification can be achieved using a
7
polymerase which is not sensitive to these substances
5 (Al-Soud et al. 2005). The same applies to the analysis of
eye fluids or muscle tissue containing myoglobin (Wiedb-
Pol
rauk et al. 1995; Bélec et al. 1998).
4
6
PCR inhibitors in food and environmental samples
Figure 1 Schematic presentation of the attack points of polymerase Many different PCR inhibitors have been identified in
chain reaction (PCR) inhibitors during sample preparation and PCR. food. Fats, glycogen, polysaccharides, minerals as well as
The nucleic acids may interfere with surfaces of the vessels (1) or sub- enzymes present in food may cause inhibition of the PCR
stances may react with nucleic acids (2) during sample processing and
(Powell et al. 1994; Richards 1999). In milk samples,
extraction. Other substances inhibit reverse transcription (3) and
degrade or modify the template DNA (4). Annealing of primers to the
PCR inhibition is mainly dependant on the concentration
template can be hampered (5) or the DNA polymerase is degraded, of calcium, whereas the fat content seems to have only
inhibited or altered (6). Finally, substances may interfere with binding minor influence on the amplification efficiency (Bickley
of probes or with their fluorophores (7). et al. 1996). Additionally, Powell et al. (1994) identified
plasmin, which degrades the Taq polymerase, as an
inhibitor naturally occurring in milk.
inhibitors and their proposed mechanisms of action are In seafood, mainly polysaccharides seem to be respon-
presented in Table 2. However, it has to be mentioned sible for PCR inhibition (Atmar et al. 1993, 1995). In
that for many inhibitory substances, the distinct mecha- addition, the glycogen content in the tissues of bivalve
nism of action is not known so far. molluscs influences PCR efficiency (Enriquez et al. 1992;
Richards 1999). Generally, the ability of bivalve molluscs
to filter the water may lead to concentration of different
PCR inhibitors in clinical samples
inhibitory substances.
In blood, serum or plasma samples, substances like IgG, Plants carry many substances, such as polysaccharides,
haemoglobin and lactoferrin have been described as polyphenols, pectin and xylan, which may be co-extracted
inhibitors of PCR (Al-Soud et al. 2000; Al-Soud and and thereafter hamper the PCR (Wei et al. 2008).
Rådström 2001). Anticoagulants, for example, heparin, Although for most of these polysaccharides (dextran, inu-
may also inhibit the PCR (Costafreda et al. 2006). Many lin, pectin or starch), no inhibitory effect was reported,
of these inhibitors are presumed to affect directly the some of them like dextran sulphate and ghatti gum affect
RNA and not the enzymes of the reaction (Konet et al. the PCR efficiency. However, this effect was partly revers-
2000). In addition, hormones or antiviral substances like ible by adding Tween 20, dimethyl sulphoxide or polyeth-
acyclovir can also affect amplification (Yedidag et al. ylene glycol 400 (Demeke and Adams 1992). In another
1996; Burkardt 2000). study, acidic polysaccharides (ghatti gum, xylan, dextran

© 2012 The Authors

1016 Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology
C. Schrader et al. PCR inhibitors

Table 2 Examples of PCR inhibitors and their mechanisms of action

Inhibitor Mechanism of action References

Polyphenols Co-precipitation with nucleic acid; reduction in John (1992), Sipahioglu et al. (2006), Su and Gibor (1988), Wan and
Polysaccharides the ability to resuspend precipitated RNA Wilkins (1994) and Wilkins and Smart (1996)
Bacterial cells Degradation/sequestration of nucleic acids Burkardt (2000), Katcher and Schwartz (1994), Peist et al. (2001),
Cell debris Rossen et al. (1992), Weyant et al. (1990) and Wilson (1997)
Detergents
PCR additives
Proteins
Polysaccharides
Salts
Solvents
Polyphenols Cross-linking with nucleic acids; change of John (1992), Opel et al. (2010) and Wilkins and Smart (1996)
Polysaccharides chemical properties of nucleic acids
Humic acids
Collagen
Melanin
Humic acid Binding/adsorption to nucleic acid and enzymes Abbaszadegan et al. (1993)
Humic matter
Haematin Incomplete melting of DNA Opel et al. (2010)
Indigo
Metal ions Reduction in specificity of primers Abbaszadegan et al. (1993)
Detergents Degradation of polymerases Powell et al. (1994), Rossen et al. (1992), Saulnier and Andremont
Proteases (1992) and Wilson (1997)
Urea
Calcium Inhibition of DNA polymerase or reverse Al-Soud et al. (2000a), Al-Soud and Rådström (1998), Eckhart et al.
Collagen transcriptase activity (2000), Opel et al. (2010), Peist et al. (2001) and Wilkins and Smart
Haematin (1996)
Herbal
metabolites
IgG
Melanin
Myoglobin
Polysaccharides
Sodium
Tannic acid
Polyphenols Chelation of metal ions Abbaszadegan et al. (1993) and Opel et al. (2010)
Tannic acid
EDTA Chelation of metal ions including Mg++ Rossen et al. (1992)
Calcium ions Competition with co-factors of the polymerase Bickley et al. (1996), Opel et al. (2010)
Antiviral Competition with nucleotides, inhibition of DNA Yedidag et al. (1996)
substances (e.g. elongation
acyclovir)
Exogenic DNA Competition with template Tamariz et al. (2006)

EDTA, ethylenediaminetetraacetic acid; PCR, polymerase chain reaction.

sulphate), polyphenols as well as inulin and pectin found This large variety leads to the presence of many different
in tea extracts were proven to inhibit PCR amplification PCR inhibitors including those already described above. In
(Peist et al. 2001). Berries are generally rich in phenols addition, dead biomass and soil may contain humic and
(e.g. anthocyanin, flavonol, ellagitannin, proanthocyani- fulminic acids, which inhibit PCR even at low
din and phenolic acids) and polysaccharides (Seeram concentrations (Ijzerman et al. 1997). In sewage sludge,
et al. 2006; Wei et al. 2008). Substances present in berries fats, proteins, polyphenols and heavy metals are found, and
and tomatoes seem to especially inhibit real-time PCR waste water contains polysaccharides, metal ions (e.g. iron
assays using TaqMan probes, whereas conventional PCR and aluminium) and RNases – all of them are common
assays are less affected (Love et al. 2008). PCR inhibitors of environmental samples (Shieh et al.
Environmental samples can be very diverse and derived 1995; Rock et al. 2010). Several different inhibitors have
from different compartments including soil, water or air. also been detected in animal feed (Löfström et al. 2004).

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Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology 1017
PCR inhibitors C. Schrader et al.

A large variety of water samples are frequently used for the

General methods for removal of PCR inhibitors
detection of pathogens. To detect even low amounts of
pathogens, large volumes of water are usually concentrated Several methods for the removal of inhibitors or for the
to very small volumes. However, this often results in con- reduction of their effects have been proposed so far and
current concentration of the different inhibitors and some examples are shown in Table 3. Generally, the
increased interference with PCR (Abbaszadegan et al. effects of the inhibitors may be reduced by selecting an
1993, 1999; Tsai et al. 1993; Jiang et al. 2005). Similar appropriate method for sample processing and nucleic
problems may occur during the collection of air samples, acid extraction, by the choice of a more robust DNA
which is usually carried out by passing large air volumes polymerase or by the use of specific PCR additives
through a variety of filters or other binding material. By (Al-Soud and Rådström 2001). For example, guanidinium
this, inhibitory airborne components are often enriched thiocyanate extraction may remove inhibitors from differ-
leading to failure of PCR (Maher et al. 2001; Chen et al. ent sample matrices more efficient than other methods
2010; Oppliger et al. 2011). (Shieh et al. 1995; Hale et al. 1996). Other effective strat-
egies include a phenol–chloroform extraction for the
removal of inhibitory lipids or a treatment with activated
PCR inhibitors introduced during sample
carbon to eliminate inhibitory salts, such as urates
preparation
(Wiedbrauk et al. 1995; Abolmaaty et al. 2007; Chaturv-
Inhibitors may be added to the sample during sample pro- edi et al. 2008). These methods are reported to be more
cessing or during nucleic acid extraction. This includes successful than gel filtration, treatment with proteinase K
powder from gloves (Demeke and Jenkins 2010), different or heat treatment (Huppertz et al. 1993; Bergallo et al.
salts (e.g. sodium chloride or potassium chloride), deter- 2006). Nevertheless, other reports show that the applica-
gents or organic molecules [ethylenediaminetetraacetic tion of the phenol–chloroform extraction was not suffi-
acid (EDTA), sarkosyl, ethanol, isopropyl alcohol or phe- cient for complete removal of PCR inhibitors (Pachner
nol] (Weyant et al. 1990; Katcher and Schwartz 1994; and Delaney 1993).
Burkardt 2000; Peist et al. 2001; Demeke and Jenkins Column chromatography using Sephacryl S-400,
2010). These substances may be necessary for efficient cell Sephadex G-200, Chelex, Sephadex or cetrimonium bro-
lysis or for the preparation of pure nucleic acids, but they mide can be used to remove salts and small proteins as
may also cause PCR inhibition at certain concentrations. well as polysaccharides from several sample matrices, for
Ionic detergents (e.g. sodium deoxycholate, sarkosyl and example, seminal fluid or stool (Da Silva et al. 1995;
SDS) are highly inhibitory for the PCR, whereas non-ionic Schmidt et al. 1995; Hale et al. 1996; Croci et al. 2008).
detergents (e.g. Nonidet P-40, Tween 20, Triton X-100 and For example, cetrimonium bromide forms an insoluble
N-octyl glucoside) cause PCR inhibition only at relatively complex with polysaccharides and denatured proteins
high concentrations (Weyant et al. 1990). EDTA is found and therefore efficiently removes them from the prepara-
in several elution buffers of purification kits for preserva- tion (Alaeddini 2011). A repeated extraction using silica
tion of DNA, but at certain concentrations, it may deplete columns may also remove inhibitors (Kemp et al. 2006).
magnesium ions and thus inhibit DNA polymerase activity. In addition, cation exchange resins have been successfully
Additives of the PCR mixture, such as dithiothreitol, used for the removal of PCR inhibitors (Jacobsen and
dimethyl sulphoxide or mercaptoethanol, may also be Rasmussen 1992; Henson and French 1993). The use of
inhibitory at certain concentrations. For some of the magnetic silica beads for nucleic acid isolation has been
polymerases commonly used in PCR, the inhibitory con- repeatedly shown to efficiently remove a wide range of
centrations of certain substances have been determined PCR inhibitors (Maher et al. 2001; Rutjes et al. 2005;
and can be retrieved from the manufacturer. Ngazoa et al. 2008; Sur et al. 2010).
Contact between polymeric surfaces and UV-irradiated Immunocapture methods are very efficient in removal
plastic tubes with PCR chemicals is reported to decrease of PCR inhibitors because the pathogen can be specifi-
the sensitivity of the PCR (Butot et al. 2007b; Fox et al. cally separated from the sample matrix and the accordant
2007; Gassilloud et al. 2007; Gonzalez et al. 2007), inhibitors (Widjojoatmodjo et al. 1992; Croci et al. 2008;
although Tamariz et al. (2006) did not find any influence Schrader et al. 2011). For example, antigen-capture PCR
of UV irradiation. However, this discrepancy might be has been successfully used for the sensitive detection of
explained by different dosages of UV light used, which hepatitis A virus in seafood (Arnal et al. 1999). However,
have been shown to correlate with the inhibitory effect because of the use of specific antibodies, this method is
(Burgess and Hall 1999). Generally, the material of swabs not applicable to highly variable pathogens, such as
or the composition of transport media may also influence noroviruses (Atmar et al. 1995). In this case, human
the sensitivity of the PCR (Wadowsky et al. 1994). histo-blood group antigens or pig gastric mucin, which

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1018 Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology
C. Schrader et al. PCR inhibitors

Table 3 Strategies for the removal of PCR inhibitors from specific matrices

Matrix Strategy for removal of inhibitors References

Stool Additional extraction steps Al-Soud and Rådström (1998), Chaturvedi et al. (2008), Hale et al.
Sedaphex G-200 chromatography (1996), Kreader (1996), Monteiro et al. (1997), Scipioni et al.
Heat treatment before the PCR (2008a,b) and Wilde et al. (1990)
BSA or gp32
Selection of resistant polymerases
Chloroform extraction
Treatment with activated carbon
Dilution of the sample
Seafood, bivalves, PEG precipitation Abolmaaty et al. (2007), Atmar et al. (1993, 1995) and Jothikumar
oysters Cetyltrimethylammonium bromide treatment et al. (2005)
Proteinase K treatment
Preparation of selected tissues (digestive gland)
Activated carbon treatment
Plants RNA precipitation Demeke and Adams (1992), John (1992), Henson and French (1993),
Treatment with: Tween 20, DMSO, PEG 400, Sipahioglu et al. (2006), Su and Gibor (1988), Wan and Wilkins
polyvinylpyrrolidone, ß-mercaptoethanol, (1994) and Wilkins and Smart (1996)
dithiothreitol
dilution of nucleic acids
cation exchanger
Extraction with high concentration of borates
Precipitation of polysaccharides
proteinase K treatment
Desiccation (65°C for 2 days)
Berries Chloroform/butanol extraction Butot et al. (2007a), Dubois et al. (2002) and Love et al. (2008)
treatment with pectinases
Use of conventional PCR instead of real-time PCR
Cheese, meat Selection of resistant polymerase Al-Soud and Rådström (1998)
Cheese Inactivation of proteases by hot NaOH extraction Rossen et al. (1992)
Milk Addition of BSA, protease inhibitors, magnesium Bickley et al. (1996) and Powell et al. (1994)
ions
Chelation of calcium ions
Water, Combined treatment of Sedaphex G-100 and Abbaszadegan et al. (1993), Cannon and Vinjé (2008), Ijzerman
environmental Chelex-100 et al. (1997) and Tamariz et al. (2006)
samples Dialysis
Extraction with solvents
Ultrafiltration with positively charged membranes,
UV irradiation
Antigen-capture PCR
Airborne and Magnetic bead DNA capture method Maher et al. (2001)
environmental
samples

BSA, bovine serum albumin; PCR, polymerase chain reaction.

are proposed to be cellular receptors for noroviruses, mixture, which may include betaine, bovine serum albu-
have been successfully used for virus capturing and min (BSA), dimethyl sulphoxide, formamide, glycerole,
subsequent detection by PCR (Cannon and Vinjé 2008; non-ionic detergents, polyethylene glycol, powdered milk,
Tian et al. 2008). T4 bacteriophage gene 32 product (gp32) and proteinase
A more general and widely applied method is the dilu- inhibitors (Frackman et al. 1998; Al-Soud and Rådström
tion of the sample or the extracted nucleic acid, which will 2000; Eckhart et al. 2000). Especially, BSA and gp32 are
automatically result in a dilution of the PCR inhibitors known to be effective against iron chloride, hemin, fulmi-
(Widjojoatmodjo et al. 1992; Monteiro et al. 1997; Eckhart nic acid, humic acid, tannic acid, stool extracts and mela-
et al. 2000; Scipioni et al. 2008a,b). However, the dilution nin (Al-Soud and Rådström 2000; Scipioni et al. 2008b;
clearly is accompanied by a decrease in sensitivity. An Opel et al. 2010). However, BSA is not effective against bile
opponent strategy is the addition of substances to the PCR salts, bilirubin, EDTA, sodium chloride, SDS, Triton

© 2012 The Authors

Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology 1019
PCR inhibitors C. Schrader et al.

X-100, calcium and collagen (Kreader 1996; Opel et al. and Smart 1996). Sipahioglu et al. (2006) removed these
2010). Other described methods such as heating of the inhibitors from leaves by drying at 65°C for 2 days and
sample or DNase treatment were not always suitable for conserving them at 4°C under hermetic conditions.
the removal of PCR inhibitors (Wilde et al. 1990). Humic and fulminic acids, which are often present in
The choice of the DNA polymerase and – if applicable – dead biomass, soil and water samples may be removed by
of the appropriate reverse transcription system is of great dialysis, liquid–liquid extraction, flocculation using poly-
importance to prevent PCR inhibitory effects valent cations, gel extraction, column-based methods and
(Al-Soud and Rådström 1998; Löfström et al. 2004). ultrafiltration, the latter one being most successful (Tsai
Kermekchiev et al. (2009) showed that distinct mutations and Olson 1992; Abbaszadegan et al. 1993; Tsai et al.
in the Taq DNA polymerase can overcome its inhibition 1993; Ijzerman et al. 1997; Braid et al. 2003). Queiroz
by blood, plasma, haemoglobin, lactoferrin, serum IgG, et al. (2001) used electropositive filters for the analysis of
soil extracts and humic acids. Baar et al. (2011) developed sewage and water samples, which preferentially bind
a chimeric polymerase consisting of parts from different microorganisms and avoid co-purification of the
polymerases of the genus Thermus, which shows a broad inhibitory substances.
resistance against a variety of organic and inorganic inhib- For the specific removal of other substances, only a
itors, as for example, humic acids, bone powder, fossilized few methods have been published. Urea present in urine
excrements, coal tar or clay-rich earths. For real-time PCR samples may be effectively removed by dialysis or ultrafil-
using TaqMan probes, the 5′ exonuclease activity of the tration (Khan et al. 1991). Proteases, which might occur
Taq polymerase is essential. In an experiment with 15 in milk, may be eliminated by the addition of protease
different Taq polymerases, considerable differences in the inhibitors or BSA (Powell et al. 1994). The inhibitory
intensity of the fluorescence in different sample types were effects caused by calcium ions may be compensated by
obvious (Kreuzer et al. 2000). the addition of magnesium ions. Another possibility is
Manufacturer of commercially available kits for nucleic the use of different chelating agents, which catch the cal-
acid purification and PCR use a variety of the above- cium ions, resulting in increased amplification rates
mentioned strategies for removal of PCR inhibitors and (Bickley et al. 1996).
increasing robustness of PCR enzymes. Several studies
investigated the performance of such kits (e.g. Ribao
Assessing PCR inhibitions by control reactions
et al. 2004; Levesque-Sergerie et al. 2007; Demeke and
Jenkins 2010); however, the efficiency is strongly depen- Despite extensive efforts to eliminate inhibitors from the
dent on the used matrix and no general recommenda- sample, inhibitory substances which can affect PCR may
tions can be retrieved from those studies. still be present. Different analytical methods can be used
to determine the presence of inorganic or organic com-
pounds, for example, the amount of dissolved organic
Methods for removal of specific PCR inhibitors
carbon indicative for humic acids (Chen et al. 2003;
Several methods have been developed for removal of spe- Rock et al. 2010). However, for most of the inhibitory
cific classes of inhibitors. Polysaccharides, which are substances, no practicable tools for their analysis are
mainly found in seafood or berries, may hamper the available.
resuspension of precipitated nucleic acids (Atmar et al. To determine the inhibitory effect of all substances
1993, 1995; Butot et al. 2007a). Either the precipitation present in a nucleic acid preparation, it has been sug-
of the polysaccharides before RNA isolation or applica- gested to carry out PCR control reactions. This is espe-
tion of an RNA isolation method without any polysac- cially necessary to exclude false-negative results, which
charide contamination is used to avoid these negative may result from complete inhibition of the PCR, even
effects (Fang et al. 1992; Wilkins and Smart 1996). This in the presence of the target sequence (Parshionikar
includes treatment with Tween 20, DMSO, polyethylene et al. 2004). Such controls generally consist of defined
glycol or activated carbon (Demeke and Adams 1992; amounts of nucleic acids or microorganisms, which are
Abolmaaty et al. 2007). Pectinase treatment has been suc- added to the sample and analysed parallel to the target
cessfully used for the analysis of berries (Butot et al. sequence. By comparison of the detected amount of
2007a). the control with that originally added, the performance
Phenols can directly interact with RNA. Removal of of the assay including the presence of PCR inhibitors
phenols can be achieved by precipitation using polyvinyl- can be assessed. Different types of controls can be dis-
pyrrolidone (John 1992; Wilkins and Smart 1996). High tinguished according to the controlled steps (process
concentrations of borates protect the RNA from interac- control, amplification control) and the implementation
tion with polyphenols (Wan and Wilkins 1994; Wilkins of the control reaction (internal or external control).

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1020 Journal of Applied Microbiology 113, 1014--1026 © 2012 The Society for Applied Microbiology
C. Schrader et al. PCR inhibitors

A process control is added at the starting point of

Conclusions
sample analysis, for example, before nucleic acid extrac-
tion, and passes therefore all preparation steps. In con- In conclusion, PCR inhibitors are a heterogeneous class of
trast, an amplification control is added to the nucleic substances that act at different steps of the diagnostic
acid extracted from the sample thus controlling only procedure. They are present in a large variety of sample
the performance of the PCR itself. An internal control types and may lead to decreased PCR sensitivity or even
is analysed in the same tube as the target, whereas the false-negative PCR results. Several strategies have been
external control is analysed in a separate aliquot of the developed to remove PCR inhibitors during sample prepa-
sample (Nolan et al. 2006). ration. However, as at least in complex matrices, it cannot
The controls can be divided into competitive and be guaranteed that the preparations are free of PCR inhibi-
non-competitive amplification controls. Competitive tors, all reactions should be analysed for the presence of
controls are amplified using the same primers and the inhibitory effects. The development of standardized con-
same conditions but can be distinguished from the tar- trols has therefore been recommended for substantiated
get by product length or sequence (Hoorfar et al. 2004; evaluation of diagnostic PCR results and for comparison of
Villanova et al. 2007). One limiting factor of this appli- the efficiencies of different PCR protocols (Hoorfar et al.
cation may be the concurrent use of primers, which 2003, 2004; Mattison et al. 2009; Lees and CEN WG6
may lead to a general decrease in PCR sensitivity TAG4 2010). On the basis of the results of such efficiency-
(Hoorfar et al. 2004). In contrast, non-competitive con- controlled assays, it might be necessary to modify the steps
trols composed of unrelated sequences are amplified by of sample analysis to remove specific PCR inhibitors or to
different primers as the target sequence and thus can use a PCR system, which is less sensitive to the inhibitory
also be used universally (Dingle et al. 2004; Hoorfar substances. This might be carried out by the application of
et al. 2004; Dreier et al. 2005; Villanova et al. 2007). A general methods for removal of PCR inhibitors, for exam-
possible disadvantage of non-competitive controls is the ple, by changing the commercially available kits for nucleic
use of separate primers, which may behave different acid preparation and PCR analysis. However, additional
than those used for the target sequence (Villanova steps for removal of specific inhibitors may be necessary.
et al. 2007). Generally, by using internal controls in The overview presented here might provide a collection of
multiplex PCR assays, the co-amplification of the con- published methods, which can be selected and tested based
trol may inhibit the amplification of the target on the sample type. In other cases, the different mecha-
sequence, which might be solved by limiting the con- nisms of action of PCR inhibitors described here might be
centration of the control primers and control RNA a starting point for the development of new methods for
(Hofmann 2003; Gall et al. 2007). their removal or inactivation.
One special kind of non-competitive control is the
endogenetic control, which uses housekeeping genes that
directly originate from the sample (Hoffmann et al. 2005). Conflict of interest
Endogenetic controls act as process controls, which control There is no conflict of interests to declare.
the whole sample processing procedure. However, a high
concentration of the endogenetic nucleic acids may hamper
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