Application of quantitative PCR for the
detection of microorganisms in water
Present the problem
- Microorganisms in water due to contamination is a health risk and control
thereof is a necessity. Conventional detection methods may be misleading and
do not provide rapid results allowing for immediate action.
- The quantitative polymerase chain reaction (qPCR) method has proven to be
an effective tool to detect and quantify microorganisms in water within a few
hours. qPCR assays have recently been developed for the detection of specific
adeno- and polyomaviruses, bacteria and protozoa in different water sources,
the technique is highly sensitive and able to detect low numbers of
microorganisms.
- Quantitative PCR can be applied for microbial source tracking in water
sources, to determine the efficiency of water and wastewater treatment plants
and act as a tool for risk assessment. Fluorescent probes are used in the PCR
reaction to hybridise within the target sequence to generate a signal and,
together with specialised systems, quantify the amount of PCR product.
This review focuses on the application of qPCR in the detection of
microorganisms in water.
- Introduction
Conventional methods used to identify microorganisms are laborious
and time-consuming and certain microorganisms are not culturable on
bacteriological media or do not exist in numbers high enough in water
to allow detection
The introduction of molecular assays has significantly improved and
simplified the detection and identification of microorganisms in the
environment. Since 1985, the polymerase chain reaction (PCR) is the
most widely used method for the amplification of nucleic acid and has
played an important role in the characterization of microorganisms
- The qPCR technique involves the
simultaneous amplification, detection and
quantification of a specific nucleic acid
target in a biological sample.
+ This is accomplished through the
monitoring of fluorescently labelled PCR
products. The methods of quantitation of
the amplicons and type of standard used
in the assay determine the strategy for
qPCR.
+ Two methods are used for qPCR,
namely end-point PCR and real-time
PCR. During end-point PCR a
measurement is taken after completion
of the entire PCR reaction to calculate
the amount of template present prior to
PCR.
+ Real-time PCR takes measurements at
the exponential phase of the PCR and is
a more sensitive and reproducible
method
Due to the fact that the direct amplification of
RNA during a PCR reaction is impossible, the
enzyme reverse transcriptase (RT) is required to
catalyse the synthesis of a complementary DNA
(cDNA) copy from the RNA template.
- This single-stranded cDNA serves as the target
during PCR amplification. This technique is
referred to as RT-PCR and is used for the
detection of enteric viruses containing RNA
genomes.
- The quantitative reverse transcription
polymerase chain reaction (q-RT-PCR) has been
used to detect and quantify enteric viruses in
environmental water sources.
- This method can also be used to measure low
levels of messenger RNA (mRNA) expression in
samples of a small volume, for the comparison of
mRNA levels in different samples,
characterization of patterns in mRNA expression
and discrimination between closely related
mRNAs
Waterborne pathogens
- Viruses - Bacteria
+ Waterborne enteric viruses include A range of bacterial pathogens are
adenoviruses, astroviruses, associated with waterborne
enteroviruses, hepatitis viruses, diseases. + The list
noroviruses, polyomaviruses and includes Salmonella typhi,
rotaviruses.
S. paratyphi,
+ Adenoviruses and polyomaviruses other Salmonella spp., Shigella spp.,
are double-stranded DNA viruses
Vibrio cholerae, E. coli spp., Yersinia
while astroviruses, hepatitis viruses,
noroviruses and rotaviruses are enterocolitica, Campylobacter
single-stranded RNA viruses. Enteric jejuni, Legionella jejuni.
viruses have been detected in + Typhoid and paratyphoid fever
different water sources including caused by S. typhi and S. paratyphi
marine, river, ground, drinking, + Epidemic cholera is caused by the
recreational and wastewater serogroups V. cholerae O1 and O39
that produce the cholera toxin
- Protozoa
Enteric pathogenic protozoa species
have been associated with the outbreak
of waterborne infections and may be
present in water due to direct or indirect
contamination with human or animal
faecal matter. Protozoa species have
several lifecycles, of which the ingestion
of either the cysts (Entamoeba
histolytica, Giardia
duodenalis and Balantidium coli) or
oocysts
(Cryptosporidium spp., Sarcocystis spp.,
Toxoplasma
gondii and Cyclospora spp.) are infective
to humans. Cysts and oocysts are highly
resistant to chlorination. Symptoms of
protozoan infections include diarrhoea
(Cryptosporidium spp., G. duodenalis, B.
coli and Cyclospora spp.), dysentery (E.
histolytica, B. coli) and fever (T. gondii).
Quantitative PCR assays
Real-time PCR
Real-time qPCR is used for gene expression quantification, expression
profiling, single nucleotide polymorphism analysis and allele
discrimination, validation of microarray data, genetically modified
organisms (GMO) testing, monitoring of viral load and other pathogen
detection applications. Different protocols exist for real-time PCR
including relative qPCR and absolute measurements.
- Relative quantitative PCR - Absolute/competitive qPCR
+ A comparison of the differences of + Absolute qPCR, also known as
nucleic acid targets in different samples competitive qPCR, is used for the
can be performed by relative qPCR. quantitation of a gene in DNA
Temporal and functional variations of measurements by comparing the levels
mRNA molecules are usually measured. observed in reference materials containing
+ The method is based on the fact that a known number of target copies of the
the difference in threshold cycles between genes. The reference material has the
the target gene and the housekeeping same prime recognition sites as the target
gene is proportional to the relative gene gene and is included in the sample as a
expression level of the target gene competitor (DNA or RNA)
+ Relative qPCR is also used for DNA + The most accurate procedure is to add
measurements where two genes are co- a known amount of the competitor to each
amplified with two primer pairs to allow assay tube to ensure the amplification of
measurement of their ratio. The reciprocal the two species at the same rate. After
variation is then determined by comparing amplification, the nucleic acid target and
with one or more control genes. Relative competitor are separated analytically to
qPCR is, however, not a sufficient method evaluate the ratio between the two
to quantify the amount or concentration of species. This final ratio reveals the ratio
nucleic acid targets as only estimated between the target and competitor in the
values are determined initial sample
Calibration standards
- To establish a PCR assay, DNA isolated from the pure culture of the
tested microorganisms should be serially diluted so that a calibration
curve can be created by the thermocycler instrument. The assay
should be repeated up to 10 times to ensure the reproducibility of the
curve and thereby to extract highly specific data
- The cycle threshold (C T) is determined automatically by the
instrument after the threshold fluorescence value is manually set. For
every assay the difference in C T of the reference sequence assay
and that of the target sequence assay is determined.
- The amplification efficiency (E) of the target assay can then be
determined by the formula . The estimated number of target
organism cells in the test sample is calculated by multiplying these
ratios by the known number of target organism cells
Multiplex real-time PCR
- Multiplex real-time PCR is used to screen large numbers of environmental
samples and to detect and discriminate different bacterial strains in a single
assay
+ The advantage over monoplex qPCR is the amplification of several DNA
targets in a single reaction that subsequently reduces cost of assays, time
and labour.
+The disadvantages include the selection of primer pairs that must function
in the same reaction conditions, the occurrence of primer dimer formation
between primer pairs leading to poor sensitivity and the preferential
amplification of certain targets that can take place.
- The assay can only be successful if the concentrations of primers, PCR
buffer, magnesium chloride vs. deoxynucleotide, cycling temperatures and
amount of DNA template and Taq DNA polymerase are correct
The following microorganisms were detected in water samples using
multiplex real-time PCR: E.
coli O157:H7 , Aeromonas spp., Salmonella spp., Shigella spp., Vibrio
cholerae, V. parahaemolyticus and Y. enterocolitica in a single assay.
Detection and measurement of PCR products
Fluorogenic probes are based on fluorescent resonance energy
transfer (FRET) systems including TaqMan, molecular beacons and
SYBR Green I, a dsDNA-binding dye.
+ Fluorogenic probes hybridise within the target sequence to generate
a signal that accumulates during PCR cycling proportional to the
concentration of amplification products. The signal is associated with
the amplified target that in turn quantifies the amount of PCR products.
+ Fluorescence can be measured at the end of PCR (end-point
fluorescence) or measured during the amplification phase (real-time
PCR).
In real-time PCR quantification takes place early, in the exponential
amplification phase of the reaction. This close-tube system requires no
post-treatment of the PCR products that reduces the changes of PCR
contamination. The real-time PCR method is therefore a more accurate
and less time-consuming method than end-point PCR.
TaqMan principle
Mechanism of the TaqMan probe.
The probes rely on the 5′–3′
nuclease activity of Taq DNA
polymerase to cleave a dual-
labeled probe during hybridisation
to the complementary target
sequence
SYBR Green I
SYBR® Green I detection
mechanism. SYBR® Green I is
1,000-fold more fluorescent in the
bound state (green star) than in
the unbound state (blue circle).
The fluorescent signal increases
proportionately as the PCR
amplification increases
Molecular beacons
Mechanism of molecular beacon
chemistry. The molecular beacon
includes a hairpin loop structure,
the loop being complementary to
a target sequence and the stem
formed by the addition of internal
complementary sequences. The
molecular beacon hybridises to
the target and the fluorophore and
quencher are far enough apart to
allow fluorescence to be detected
Use of qPCR for the detection and
quantification of microorganisms in water
DNA can be extracted by a variety of DNA kits in combination
with bead beating depending on the tested microorganism.
Cell lysates can also successfully be used with the same
reproducibility of purified DNA and involves less labour. The
genomic segment(s) to be amplified is (are) then selected and
the optical tubes containing the reaction mixtures are
subjected to thermal cycles that vary between 1 h and 15 min
to 2 h and 45 min. The amplified genomic segment(s) is (are)
then detected and quantified by using specific protocols
(Tables 1 and 2). To determine the presence of inhibitors, the
tested water sample is autoclaved and a pure culture of the
test organism is cultured and inoculated into the water sample
