Download HLA-B27 real-time PCR using TaqMan

HLA-B27 real-time PCR using TaqMan-MGB
sequence specific probes
Stewart M Smith, Andrew D Laurie, Howard C Potter and Ben D McGettigan
Abstract
Objectives: The HLA-B*27 genotype is strongly associated
with ankylosing spondylitis (AS) and also other
spondyloarthropathies (SpA) such as psoriatic SpA. Our
objective was to develop and validate a real time PCR for HLAB*27 with greater capacity than our current allele specific
approach without loss of fidelity.
Methods: We describe a real-time PCR method for the
detection of HLA-B*27 using allele specific primers combined
with TaqMan fluorescent minor groove binder (MGB) probes.
One probe is specific for human leucocyte antigen (HLA) B*27
conjugated with the fluorochrome FAM and the other specific
for human growth hormone (HGH) conjugated with the
fluorochrome VIC which acts as endogenous control.
Results: 441 consecutive samples were tested of which 139
tested HLA-B*27 positive and 302 tested negative. We tested a
further 12 external quality control samples of which four were
positive and eight negative for HLA-B*27.
Conclusions: RT-PCR is suitable for HLA-B*27 genotyping
and has the advantages of lesser hands-on time, does not
require any post amplification processing, and does not use the
toxic DNA intercalating dye ethidium bromide.
Keywords: HLA-B*27, TaqMan, PCR, MGB, minor groove
binder, NFQ, non-fluorescent quencher, real time PCR,
ankylosing spondylitis.
N Z J Med Lab Sci 2014; 68: Introduction
The human leucocyte antigen (HLA) B*27 is strongly associated
with the spondyloarthropathies (SpA’s). HLA-B*27 is found in
90-95% of ankylosing spondylitis (AS) patients and also lesser
frequency in forms of psoriatic arthritis and reactive arthritis
(1,2). HLA-B*27 may have a direct role in the pathogenesis of
SpA which is supported by epidemiological studies and
transgenic rat studies (3). Different hypotheses exist, such as
the arthritogenic peptide hypothesis, the misfolding hypothesis
or molecular mimicry (3-5). Less than 5% of those members of
the general population that are HLA-B*27+ develop AS, and
HLA-B*27 has been estimated to account for only 20-50% of
the overall genetic susceptibility to AS (6). In addition to HLAB*27, a recent genome wide association study has confirmed
25 loci that confer increased genetic risk for AS, including
genes involved in peptide processing prior to MHC class I
presentation and alterations in the IL-23 pathway (7).
The aim of our study was to adapt our conventional allele
specific HLA-B27 protocol to a fluorogenic real time (RT-PCR)
method. The allele specific PCR detects the presence of the
B*27 genotype by amplifying a region between primer sets that
recognise only B*27 specific sequences. The PCR reaction
amplifies this region to sufficient quantity so that it may be
visualised after electrophoresis in agarose gel. Visualisation is
achieved by staining with ethidium bromide (a fluorescent DNA
intercalating dye) and viewing the gel under UV light (Figure 2).
Both allele specific and RT-PCR methods detect the presence
or absence of B*27 and do not discriminate between
heterozygous and homozygous individuals. The RT-PCR
method involves the addition of specific dual labelled hydrolysis
probes to the PCR. These TaqMan probes have a fluorescent
reporter (ie. FAM or VIC) attached to the 5ƍ end of the probe
sequence and a non-fluorescent quencher (NFQ) attached to
the 3ƍ end. Addition of a minor groove binder (MGB) to the
probe sequence at the 3ƍ end of the probe permits it to anneal
at a higher melting temperature (Tm), allowing the use of
shorter more specific probes (2,18). The probe in free form is
normally quenched as random coiling brings the probe and
fluorophore close together (19). The MGB works together with
the quencher to improve quenching. During the extension step
of the PCR, the probe binds to its complementary sequence at
a position that sits between the forward and reverse primers.
(Fig 1) During extension Taq polymerase cleaves the probe,
because of intrinsic 5ƍ to 3ƍ nuclease activity, releasing the
fluorescent reporter (FAM or VIC) into the mix thus causing an
irreversible increase in fluorescence(19). As the cycles continue
fluorescence increases rapidly until the probe is exhausted. The
advantages of this method are that primers and probes provide
specificity to the reaction products and allow immediate
visualisation. It is possible to use several different probes in
each PCR to simultaneously detect specific products. In this
case both the HLA-B*27 and HGH (internal control) products
are detected independently. The analytical capacity of this
technique is much greater than the allele specific PCR and
allows for near complete automation.
Figure 1A. Target DNA, primers and probes are mixed
together.
There are more than 50 subtypes of HLA-B27 identified which
mostly differ by only a small number of nucleotides. (IMGT/HLA
database: http:\\www.ebi.ac.uk/imgt/hla/). The subtypes HLAB*27:02, B*27:04, B*27:05, B*27:07, B*2:708 are associated
with AS although B*27:07 is suggested to be associated but not
in Greek Cypriots (8,9). Important conformational differences
exist in subtypes not associated with AS (4,10-14).
Many laboratories use an allele specific PCR based on that of
Sayer et al (17), which offers excellent specificity for HLA-B*27
although they report a lack of sensitivity for subtype HLAB*27:12. This lack of sensitivity is also likely for the subtypes,
HLA-B*27:16, HLA-B*27:18, HLA-B*27:23, HLA-B*27:29.
Figure 1B. After melting, primers and probes are allowed to
bind to their complementary sequences.
NZ J Med Lab Science 2014
4
One cycle of denaturation at 96°C for 4 minutes was
followed by 30 cycles of 96°C for 30 seconds, 66°C for 2
minutes, 72°C for 30 seconds followed by a final extension at
72°C for 5 minutes. Amplified products were visualised under
UV light after electrophoresis on a 2% agarose gel and staining
with ethidium bromide.
Figure 1C. Taq Polymerase extends primers and in the process
its nuclease activity removes probes causing an irreversible
increase in fluorescence in the process.
MGB: minor groove binder. NFQ: non-fluorescent quencher.
Pol: polymerase. FAM: fluorescent dye (6-Carboxyfluoroscein).
Materials and methods
Sample DNA
DNA was isolated from EDTA whole blood samples from 441
consecutive sample sent for HLA-B*27 testing at our laboratory
with a TECAN Genesis RSP 100 liquid handling system, using
the AGOWA® mag Maxi DNA Isolation Kit.
An additional 12 external control samples from the Royal
College of Pathologists of Australasia Quality Assurance
Programs Pty Ltd (RCPA QAP) HLA-B*27 program were
prepared. The average DNA concentration as measured by
NanoDrop ND-1000 ver 3.1.2 software, National Instrument
Corporation, was 20ng/μl with the 5th to 95th percentile being
11 – 34 ng/μl. A 5μl aliquot of DNA was used for each PCR
reaction, and therefore approximately 100ng of each sample
was used, although there was considerable variation.
Real time PCR
Allele specific PCR primers were used as above, according to
the method of Sayer et al (17). The Alt HGH internal control
primers / HGHVIC1 probe used were different from the ones
above and they were designed using ABI Primer Express v3.0
software (Table 1). The same B27 primers above were used
combined with a 22 nt. fluorescent TaqMan probe B27FAM1,
which was designed manually in a configuration allowing
efficient binding between the B27EX294F / B27EX2199RC
primers (Table 1). Both TaqMan probes were purchased from
Applied Biosystems while primers were purchased from Sigma.
Probe concentrations of 0.25μM and Primer concentrations of
0.5μM were used in PCR reactions. Samples were then
amplified using the Roche Lightcycler 480 in 15μl reaction
volumes using 10μl of PCR buffer (Roche Lightcycler 480
Probes master mix), primer/probe mix and 5μl of undiluted
DNA. One cycle of denaturation at 95°C for 5 minutes was
followed by 40 cycles of 95°C for 15 seconds, 66°C for 15
seconds, and 72°C for 30 seconds. The intensity of
fluorescence of the two different fluorophores was measured
each cycle as the PCR proceeded in real time. Samples are
considered positive when fluorescence intensity rose above the
threshold value, which usually occurs from cycle 26 to 30.
Table 1. Primers and TaqMan probes used in the RT-PCR HLAB*27 genotyping procedure.
B7 positive control samples
Four of the HLA-B*27 external quality assurance samples
provided by RCPA Immunology QAP were reported as
heterozygote for HLA-B*7 and negative for HLA-B*27. These
samples were included as part of the validation to provide
assurance of primer specificity.
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Allele specific PCR
Allele specific PCR was carried out according to the method of
Sayer et al (17). Briefly, sequence-specific primers amplify a
141bp band from exon 2 of the HLA-B*27 family. Internal PCR
amplification control primers specific for the human growth
hormone (HGH) are used for each sample to ensure there is
sufficient sample DNA for amplification to proceed or to show
amplification failures for any other reason. This internal control
produces a 437bp band which must be present to validate the
assay. The PCR was performed in a total volume of 25μl
containing 2.5μl of 10x Roche FastStart PCR Buffer with
2mM MgCl2, 2.5μl of 2mM dNTP’s, 5.0μl of 5x FastStart GC
rich buffer, 1U FastStart Taq polymerase. The reactions
contained 560nM B*27 specific primers and 280nM HGH
specific primers along with 5.0μl of 1/5 diluted DNA from each
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F\FOH RI PCR and the signal from each probe was
separated by its distinct emission spectra collected using
appropriate filter sets. Curves were then constructed with Cycle
number (x-axis) vs log fluorescence intensity (y-axis) for
each well. Fluorescence intensity for the B*27 positive
samples was usually about 15 times that of negative
controls and blanks by cycles 35 to 40. When the
fluorescence level rose above the discriminator (horizontal
dashed line) the sample was scored as positive for that probe.
Figure 2. Conventional allele specific PCR.
Reverse black/white image of typical 2% agarose gel showing
437bp human growth hormone specific band and positive
samples also displaying the 141bp B*27 specific band. Samples
amplified with conventional allele specific PCR.
NZ J Med Lab Science 2014
5
from 123ng to 246ng there was no difference in the cycle
number threshold suggesting saturation of amplification at
higher concentrations. The sensitivity of detection was therefore
at least 0.12ng/μl. This is well below the lowest concentration of
DNA isolated by our DNA isolation system by a factor of at least
10.
Figure 3. A curve for each well is constructed for HGH (Figure
3A) and HLA-B*27 (Figure 3B) specific probes with cycle
number (x-axis) vs. log fluorescence intensity (y-axis). The
discriminator (horizontal dashed line) is set manually and
results calculated for each well. Positivity is expressed as a red
square for those wells where fluorescence levels are above the
threshold.
Results
Of the 453 comparative samples tested, 100% concordance
with the conventional allele specific PCR was recorded.
Table 2. Comparison of results between allele specific and RTPCR.
Allele Specific
PCRRT-PCR
HLA-B*27 positive
HLA-B*27 negative
RCPA QAP HLA-B*27 positive
RCPA QAP HLA-B*27 negative
139
302
4
8*
139
302
4
8*
Total
453
453
*Four of the HLA-B*27 external quality assurance samples
provided by the RCPA Immunology QAP program were
reported as heterozygote for HLA-B*7.
Effects of DNA Dilution
Five HLA-B*27 positive and two HLA-B*27 negative samples
were chosen to perform dilution experiments. These samples
were all extracted as described previously and had DNA
concentrations ranging from 15 – 65ng/μl. Each sample was
serially diluted eight times, with dH2O so as to obtain a halving
of DNA in each successive dilution and these samples were
then analysed by the RT-PCR assay. This gave a range of DNA
concentrations over which the sensitivity of this test could be
measured, from 0.12 – 65ng/μl. Ideally for each doubling
dilution of DNA one would expect to see a one cycle increase in
the cycle number at which fluorescence levels increase above
an arbitrary threshold. This would hold true so long as the
threshold is set in the exponential phase of amplification.
The question of sensitivity of detection can be answered by
observing the lowest concentration at which fluorescence is
clearly distinguishable from negative samples and blank
reactions containing no DNA. Figures 4A and 4B below show
how reactions proceeded for both the HGH and B*27 specific
fluorescent probes. Fluorescence increased exponentially until
buffer constituents were exhausted whereupon the
fluorescence plateaus. The figures show that positive signals
were obtained over the full range of concentrations used. Of
note, Figure 4B shows that when DNA concentration increased
Figure 4. VIC fluorescence from HGH (Figure 4A) and FAM
fluorescence from B*27 (Figure 4B) specific probes.
Fluorescence levels cross the threshold on average one cycle
later for each halving of input DNA .
Fluorescence intensity for the B*27 specific probe reached the
plateau level in all dilutions for all five samples tested (data not
shown). Fluorescence intensity for the HGH specific probe did
not reach plateau levels in all dilutions and Figure 4A is typical
of the seven samples tested.
By graphing the cycle number value against DNA
concentration, all the samples tested can be represented with
clarity that would be lost if every fluorescence curve was
represented as in Figure 4. Figures 5A and 5B show a
relatively linear decrease in cycle number (average 1.0 – 1.1
cycles) required to reach the exponential phase of amplification
as a function of halving DNA concentration. Samples 2, 3, and
4 departed from this linearity at higher DNA concentrations for
the B*27 probe. Sample 2 also departed significantly at lower
concentrations and was observed to cross the threshold line 2.2
cycles later than the next highest dilution for the B*27 probe
rather than 1. This demonstrates that wide ranges of DNA
concentrations can be used in the RT-PCR, however, at the
extremes of DNA concentration measured the reaction
efficiency is not optimal.
NZ J Med Lab Science 2014
6
HLA-B*27:18, HLA-B*27:23 and HLA-B*27:29 have altered B
pockets because of a substitution at residue 67 (Ser, Phe and
Ser respectively instead of Cys (6,23,24). This mutation in
transgenic rats leads to gut disease but markedly less
susceptibility to arthritis (25). HLA-B*27:16, HLA-B*27:18, HLAB*27:23 and HLA-B*27:29 as well as HLA-B*27:12 all share
Thr/Asn/Thr at residues 69/70/71 instead of Ala/Lys/Ala shared
by other B*27 (23,24). This substitution is within the B pocket
and for these rare subtypes disease association with AS is not
established.
A number of subtypes contain single nucleotide mismatches
with one of the primers used in this study, B27EX2199RC. The
effect of primer-template mismatches has been studied by
others such as Kwok et al (26). They found “… that a single
mismatch 3 residues from the 3ƍ terminal base of a primer can
be efficiently extended without modification of amplification
reactions.” Wu et al (27) however “… observed that the single
mismatches present at the last five positions from the primer 3ƍ
end could totally block the primer extension.” The one base pair
mismatches in the other rarer subtypes HLA-B*27:26, HLAB*27:31 and HLA-B*27:39 are unlikely to prevent amplification
as they occur at positions 9 to 16 from the 3ƍ end of the primer.
However, the one base pair mismatch for the rarer subtype HLA
-B*27:0509 occurs at position 5 from the 3ƍ end of the primer,
which may interfere with binding according to Wu et al (27).
Primer B27EX2199RC has been observed to correctly amplify
up to an annealing temperature of 69°C (data not shown) and
as our RT-PCR assay routinely anneals 3°C lower, at 66°C, this
may give the primer some mismatch tolerance.
Figure 5. Total DNA per PCR reaction (x-axis) vs. cycle
number (y-axis) for HGH specific probe (A) and B*27 specific
probe (B). Each line represents a single sample and is
constructed by plotting the cycle at which fluorescence first
crosses the threshold line at each DNA dilution. The average
crossing time (Ct) increase is between 1.0 and 1.1 cycles.
Discussion
We have shown that a RT-PCR for the detection of HLA-B*27
can be performed that gives 100% concordance with our
current allele specific PCR. Furthermore it also showed 100%
agreement with external control samples. The method showed
no cross reactivity with the four HLA-B*7 positive samples
available. DNA concentration did not affect interpretation of this
test when DNA is prepared by our automated DNA extraction
method. The method sensitivity to DNA concentration was well
within the range isolated routinely in our laboratory. There were
signs of departure from linearity for some samples at higher
DNA concentrations and for one sample at low concentrations
which did not affect the ability to discriminate whether the
samples were positive or negative.
Other investigators have shown applications using a Lightcycler
PCR thermocycler such as Sylvain et al (2) and Bon et al ( 20).
Bon et al (20) used SYBR green I and melting curve analysis
for genotyping of HLA-B27 and Sylvain et al (2) used a TaqMan
-PCR assay using sequence-specific primers and a MGB
fluorescent probe with the use of an artificial exogenous internal
positive control (IPC) DNA added to each PCR reaction. This
was used to distinguish true negatives from false negatives.
Our assay is advantageous in that it does not use artificial
exogenous control material, but instead the HGH gene as the
internal control. This is an important distinction as both the
quantity and quality of DNA isolated, including the presence of
any PCR inhibitors, is reflected by the HGH internal control.
This is something that exogenous control material cannot do.
Of note Sayer DC et al (17) reported a lack of sensitivity for
HLA-B*27:12 because of a different B pocket configuration in its
peptide binding groove and differs from HLA-B*27:05 AS
associated F pocket configuration. For these reasons it is
unlikely to be associated with AS(17,21), although it may be
associated with reactive arthritis (22).
In conclusion, this real time PCR reliably and accurately types
whole blood samples for HLA-B27. It is very easy to automate
using the AGOWA® mag Maxi DNA isolation kit for extraction of
DNA through to end result as there is no post PCR gel
electrophoresis and photographic interpretation. This minimises
contamination risk of post amplification PCR products and
overcomes the use of the DNA intercalating dye and known
carcinogen ethidium bromide. This technique is suitable for HLA
-B*27 genotyping in the routine laboratory.
Acknowledgements
This work was supported by the Immunology and Molecular
Pathology sections at Canterbury Health Laboratories.
Author information
Stewart Smith, DipMLT, Medical Laboratory Scientist1
Andrew D Laurie, PhD, Scientific Officer2
Howard C Potter, MSc FNZIMLS MHGSA, Acting Section
Head2
Ben D McGettigan, MB ChB, Registrar1
Immunology Laboratory and 2Molecular Pathology Laboratory,
Canterbury Health Laboratories, Canterbury District Health
Board, Christchurch,New Zealand.
1
Corresponding author: Stewart Smith, Immunology,
Canterbury Health Laboratories, PO Box 151, Christchurch,
New Zealand. Email: [email protected]
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