JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2005, p. 1256–1260 Vol. 43, No.

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Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Multiplex Real-Time PCR Assay Using Scorpion Probes

and DNA Capture for Genotype-Specific
Detection of Giardia lamblia on
Fecal Samples
Cherie T. Ng,1 Carol A. Gilchrist,1 Ariel Lane,1† Shantanu Roy,2
Rashidul Haque,2 and Eric R. Houpt1*
Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia,1
and Centre for Health and Population Research, International Centre for Diarrhoeal
Disease Research, Bangladesh, Dhaka, Bangladesh2
Received 1 August 2004/Returned for modification 14 October 2004/Accepted 9 November 2004

Two major genotypic assemblages of Giardia lamblia infect humans; the epidemiologic significance of this
phenomenon is poorly understood. We developed a single-vessel multiplex real-time PCR (qPCR) assay that
genotypes Giardia infections into assemblages A and/or B directly from fecal samples. The assay utilized
Scorpion probes that combined genotype-specific primers and probes for the 18S rRNA gene into the same
molecule. The protocol was capable of detecting as few as 20 trophozoites per PCR on fecal DNA isolated using
a commercial method or 1.25 trophozoites per PCR on fecal DNA isolated using a G. lamblia-specific oligo-
nucleotide capture technique. The assay was specific for fecal specimens, with no amplification of the discor-
dant genotype with the opposite Scorpion probe. When 97 clinical specimens from Bangladesh were used, the
multiplex PCR assay detected 95% (21 of 22) of Giardia microscopy-positive specimens and 18% (13 of 74) of
microscopy-negative specimens. Microscopy-negative and qPCR-positive specimens had higher average cycle
threshold values than microscopy-positive and qPCR-positive specimens, suggesting that they represented true
low-burden infections. Most (32 of 35) infections were assemblage B infections. This single-reaction multiplex
qPCR assay distinguishes assemblage A Giardia infections from assemblage B infections directly on fecal samples
and may aid epidemiologic investigation.

Members of the genus Giardia are the most commonly di-
agnosed intestinal parasites in the United States and cause
approximately 200 million clinical infections per year world-
wide (7). The genus can be distinguished on the basis of mor-
phology, ultrastructural features, or 18S rRNA sequence into
at least six species, G. lamblia (synonymous with G. duodenalis
or G. intestinalis), G. agilis, G. muris, G. ardeae, G. psittaci, and
G. microti (17). Isolates of G. lamblia have been further sub-
grouped by alloenzyme or sequence analysis of the glutamate
dehydrogenase, triose phosphate isomerase, elongation factor
1␣, 18S rRNA, and other genes. Depending on the assay, G.
lamblia subgroup nomenclature has included Nash groups 1 to
3 (19), genotype “Poland” versus “Belgium” (12), and assem-
blages A and B with subgroups A-I, A-II, B-III, and B-IV (16).
Despite the complicated terminology, phylogenetic sequence
analysis of the independent genetic loci has provided essen-
tially concordant results showing that there are two major G.
lamblia groups which cause human infection (14, 17). Use of
the assemblage A and B nomenclature for these two main
groups has been proposed, and we will adopt it hereafter for
convenience.
While the genotypic separation of G. lamblia assemblages is
* Corresponding author. Mailing address: Division of Infectious
Diseases and International Health, University of Virginia, MR4 Build-
ing, Room 2144, P.O. Box 801340, Charlottesville, VA 22908. Phone:
(434) 243-9326. Fax: (434) 924-0075. E-mail: erh6k@virginia.edu.
† Present address: Spelman University, Atlanta, GA 30314.
relatively well established, the clinical or epidemiologic signif-
icance of infection with assemblage A versus B is poorly un-
derstood. One report from Mexico (6) indicated that 11 iso-
lates were assemblage A type, while another from Canada
indicated that 12 of 15 clinical specimens were assemblage B
type (9). Similarly small studies from India and Australia re-
sulted in reports that assemblage A was associated with symp-
tomatic infection relative to assemblage B (20, 22). In contrast,
a correlation between assemblage B and persistent diarrheal
symptoms was observed in The Netherlands (11).
More complete data on the epidemiology of infection with
individual Giardia genotypes may enhance the clinical signifi-
cance of detection and aid outbreak investigation. Existing
diagnostic methods for use on human feces such as microscopy
and enzyme-linked immunosorbent assay (ELISA) do not dis-
criminate between the two assemblages. Genotype-specific
PCR or PCR-restriction fragment length polymorphism has
been performed by several investigators (3, 5, 10, 18) but is a
time-consuming two-step procedure subject to contamination.
Several real-time PCR (qPCR) assays for Giardia have subse-
quently been published (4, 8, 26, 27), including three that are
capable of genotyping (2, 9, 13); however, most require mul-
tiple reactions, and details on assay sensitivity are unclear.
Lastly, an assay using multiplex PCR and microarray hybrid-
ization has been published; however, that assay used only cul-
tured trophozoites (28). We therefore sought to develop a
qPCR assay utilizing self-probing amplicon primers (30) that
would distinguish assemblages A and B in a single reaction and

1256
VOL. 43, 2005 GENOTYPE-SPECIFIC qPCR FOR GIARDIA LAMBLIA 1257

that was sufficiently sensitive for high-throughput use directly

on human fecal samples.
MATERIALS AND METHODS
Spiking of fecal samples. Giardia lamblia lines WB and GS (assemblage
A-group 1 and assemblage B-group 3, respectively; provided by Steven Singer,
Georgetown University, and Rodney Adam, University of Arizona) were cul-
tured axenically in TY-S-33 medium at 37°C. Trophozoites were counted in a
hemocytometer, sedimented, washed in sterile phosphate-buffered saline (PBS),
and spiked into 200-mg aliquots of parasite-free stool as indicated.
Human fecal specimens. Stool specimens were obtained from individuals with
diarrhea at the International Centre for Diarrheal Diseases and Research,
Dhaka, Bangladesh. Informed consent was obtained from the parents or guard-
ians of all participants, and the human experimentation guidelines of the U.S.
Department of Health and Human Services, the University of Virginia, and the
Centre for Health and Population Research, International Centre for Diarrheal
Disease Research, Bangladesh, were followed in the conduct of this research. All
specimens were tested for Giardia infection by saline wet-mount microscopy
after staining with Lugol’s iodine. PCR-positive specimens were additionally
tested using a Giardia II kit (Techlab, Blacksburg, Va.) for detection of Giardia
antigen per the manufacturer’s instructions.
DNA extraction. DNA was extracted from spiked stool samples and human
fecal specimens both by the commercial QIAamp method and by DNA capture.
For the QIAamp method, fecal specimens were washed twice with sterile PBS
and centrifuged for 5 min at 18,000 ⫻ g; the fecal pellet was then subjected to six
cycles of freeze-thaw in liquid nitrogen and a 95°C water bath. DNA was then
extracted using a QIAamp DNA Stool Mini kit (QIAGEN, Valencia, Calif.) per
the manufacturer’s instructions except that the suspension was incubated in the
kit’s stool lysis buffer at 95°C and a 3-min incubation with InhibitEx tablets was
performed. Genomic DNA of G. lamblia, Entamoeba histolytica, E. dispar, E.
moshkovskii, and Cryptosporidium parvum was obtained by this method using
pure trophozoite or oocyst pellets. For sequence-specific DNA capture, fecal
samples were processed according to the protocol of Shuber et al. (23). Briefly,
200 mg of spiked stool was homogenized in 1.4 ml of EXACT Buffer A (Exact
Sciences Corporation, Marlborough, Mass.) and centrifuged twice for 20 min at
18,000 ⫻ g. The supernatant was treated with 10 ng of RNase and DNA precip-
itated with 300 mM sodium acetate and isopropanol. DNA was then resuspended
in 200 ␮l of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4), incubated at 95°C for
10 min, cooled on ice, and then incubated at 25°C for 4 h in an equal volume of
6 M guanidine thiocyanate and 200 pmol of biotinylated oligonucleotide 5⬘-GC
TAGCCGGACACCGCTGGCAAC-3⬘, a sequence that is common to both as- FIG. 1. Sequence and mechanism of Scorpion probes for detection
semblage A and B (see accession numbers below) and is downstream from the of the 18S rRNA gene of assemblage A and B Giardia lamblia. (A) 18S
PCR amplicon (Fig. 1A). This suspension was then incubated for 1 h at room rRNA gene sequence indicating the assemblage-specific Scorpion
temperature with streptavidin-coated MagneSphere Paramagnetic Particles probe primer sites (boxed) and tail sites that bind to the neosynthe-
(Promega, Madison, Wis.). Bead-DNA complexes were washed three times in sized strand (underlined), the common reverse primer site (boxed),
buffer (1 M NaCl, 0.01 M Tris-HCl, 1 mM EDTA, 0.1% Tween 20, pH ⫽ 7.4) and the sequence of the biotinylated oligonucleotide used to capture
and incubated at 85°C for 10 min in 50 ␮l of 10 mM Tris (pH ⫽ 7.0). DNA from fecal specimens (shaded). (B and C) A schematic repre-
Oligonucleotides. We reviewed the literature that grouped human G. lamblia sentation of the binding of Scorpion probes during annealing and
isolates by 18S rRNA sequence into assemblages A and B (25), assemblage A extension is shown for the assemblage A- and B-specific Scorpion
and B subgroups I to IV (17), and groups 1 to 3 (29) and obtained the following probes (B and C, respectively). During extension the Scorpion probe’s
sequences from the National Center for Biotechnology Information database: stem-loop unfolds to bind the neosynthesized strand, dissociating the
for assemblage A, AF199446, M54878, X52949, and AY130269 to AY130281; for HEX or FAM fluorophore from the quencher (BHQ1). Assemblage-
assemblage B, AF113897, AF113898, AF199447, and U09492, which encompass specific sequences utilized by these Scorpion probes are shown boxed
subgroups A-I (M54878), A-II (X52949), B-III (AF113897), and B-IV (AF113898). (B and C). HEG, hexethylene glycol reverse-extension blocker. The G.
Sequences were aligned using Clustal X version 1.8 (24; http://www.ebi.ac.uk lamblia 18S rRNA sequence is numbered per accession number
/clustalw/). Assemblage A-specific forward primer AF (5⬘-ATCCTGCCGGAG U09492 (assemblage B).
CGCGACG-3⬘) and assemblage B-specific forward primer BF (5⬘-CGGTCGA
TCCTGCCGGAATC-3⬘) (similar to those used by Johnson et al.) (13) were
paired with the reverse primer R3 (5⬘-GGGGTGCAACCGTTGTCCT-3⬘)
(common to both assemblage A and B sequences) to produce 95- and 102-bp strain, M54878); however, detection of groups 2 and 3 cannot be determined,
products, respectively (Fig. 1A). Primers demonstrated no deleterious secondary insofar as the group 2 and 3 18S rRNA sequences described by Weiss et al. are
structures, and no significant identity (E ⬍ 1.0) to non-Giardia sequences was short (⬃183-bp) fragments downstream from and not inclusive of our amplicon
found by a BLAST search (1; http://www.ncbi.nlm.nih.gov/BLAST/). Scorpion (29).
Uni-probes (Proligo, Paris, France) specific for assemblage A and assemblage B PCR amplification. Singleplex amplifications took place in 25-␮l reaction
sequences were designed, whereby a 5⬘ reporter dye, a specific stem-loop se- mixtures containing 1⫻ PCR buffer (QIAGEN), 3.5 mM total MgCl2 (including
quence, a black-hole quencher (BHQ1), and a hexethylene glycol (HEG) re- that contained in the PCR buffer), 0.5 mM (each) deoxynucleoside triphosphates
verse-extension blocker were linked to the above-mentioned forward primers as (dNTPs), 1.25 U of Taq polymerase (HotStarTaq; QIAGEN), 6% dimethyl
follows: for ScA, hexachloro-6-carboxyfluorescein (HEX)CCCGGCGCATGGC sulfoxide (DMSO), 50 ␮g of bovine serum albumin (BSA), 7.44 pmols of Scor-
TTCGTCCTTGCCGGG-BHQ1-HEG-ATCCTGCCGGAGCGCGACG; for pion probe and reverse primer, and 5 ␮l of fecally extracted DNA. Multiplex
ScB, 6-carboxyfluorescein (FAM)-CGGGCATGCATGGCCCG-BHQ1-HEG-C reactions were performed in 25-␮l volumes with 1⫻ PCR buffer, 4.0 mM total
GGTCGATCCTGCCGGAATC. Underlined sequences indicate the regions of MgCl2 (including that contained in the PCR buffer), 1.0 mM dNTPs, 2.5 U of
the stem-loop sequence that are complementary to the neosynthesized strand Taq polymerase (HotStarTaq; QIAGEN), 6% DMSO, 50 ␮g of BSA, 7.44 pmols
(Fig. 1B and C). Of note, ScA aligns with Nash group 1 sequence (the Portland-1 of each Scorpion probe and reverse primer, and 5 ␮l of fecally extracted DNA.
1258 NG ET AL.
FIG. 2. Sensitivity of qPCR assay for G. lamblia assemblages A and
B. Dilutions of assemblage A or B trophozoites were spiked into
parasite-free stool, DNA was extracted using either the QIAamp (solid
lines) or oligonucleotide-capture (hatched lines) method, and single-
plex qPCR amplification was performed using Scorpion probes specific
for assemblage A (A) or assemblage B (B). Legend values indicate
numbers of spiked trophozoites per PCR. No fluorescence was ob-
served with stool samples spiked with up to 20,000 trophozoites of the
opposing assemblage/reaction or with unspiked stool (data not shown).
Horizontal line indicates fluorescence threshold.
Amplification was performed on an iCycler (Bio-Rad, Hercules, Calif.) under the
following cycling conditions: 95°C for 15 min and then 40 cycles of 94°C for 20 s
and 60°C for 30 s. Amplification was confirmed in all reactions by gel electro-
phoresis; therefore, a 5-min 72°C incubation was added at the end of the cycling
protocol.
Statistics. Values for the average threshold of qPCR amplification (cycle
threshold [CT]) were compared between microscopy-positive and microscopy-
negative groups by the Mann-Whitney test. The nonparametric Spearman cor-
relation was used to quantitate the degree of association between ELISA optical
density and CT on ELISA-positive and qPCR-positive specimens.
RESULTS
Sensitivity of singleplex qPCR. The sensitivity of the assay
using the QIAamp extraction method to detect G. lamblia
DNA in stool was ⱕ20 trophozoites per reaction for assem-
blage A (CT ⫽ 37.4) and ⱕ200 trophozoites per reaction for
assemblage B (CT ⫽ 35.0) (Fig. 2). Sensitivity was increased
with the addition of BSA and DMSO and was not enhanced by
dilution of the DNA samples (data not shown), suggesting that
the DNA yield was limiting as opposed to PCR inhibition. We
therefore utilized a DNA extraction method involving se-
quence-specific gene capture of a downstream region of the
J. CLIN. MICROBIOL.
18S rRNA gene conserved between assemblages (Fig. 1A).
DNA obtained by this method amplified at least 16-fold more
efficiently than the QIAamp extraction procedure (equal or
greater fluorescence at 1.25 versus 20 trophozoites/reaction for
assemblage A and 12.5 versus 200 trophozoites/reaction for
assemblage B; Fig. 1).
Specificity of singleplex qPCR. The specificity of the assem-
blage A- and B-specific Scorpion probes was tested on genomic
DNA from G. lamblia assemblages A and B as well as on DNA
isolated from stool samples spiked with up to 20,000 assem-
blage A or B trophozoites per reaction. No amplification was
detected by qPCR or gel electrophoresis on discordantly
spiked stool samples, regardless of parasite concentration or
the method of DNA extraction (data not shown). Amplifica-
tion of the discordant G. lamblia assemblage was only observed
when pure genomic DNA (9.9 ⫻ 105 trophozoites/reaction)
was used; such amplification was extremely inefficient, yielding
a CT equivalent to that obtained using 30 trophozoites of the
concordant assemblage/reaction (data not shown). No ampli-
fication was observed with either Scorpion probe upon using
genomic DNA of E. histolytica, E. dispar, E. moshkovskii, or
Cryptosporidium parvum (data not shown).
Multiplex PCR. The two singleplex assays were combined to
create a single-tube multiplex assay for use with human stool
samples (Fig. 3). The test was specific, with no FAM fluores-
cence observed with assemblage A-spiked fecal samples (Fig.
3A) and no HEX fluorescence observed using B-spiked fecal
samples (Fig. 3B). Sensitivity remained at the level of the
singleplex assays after optimization for magnesium, dNTP, and
Taq polymerase concentrations per procedures described
above in Materials and Methods, and CT levels were not di-
minished in coinfected versus monoinfected specimens (Fig.
3C versus 3A or 3B).
Comparison of multiplex PCR with microscopy for human
clinical specimens. Samples of 97 human stool specimens from
diarrheal patients at the International Centre for Diarrheal
Diseases, Bangladesh, were tested by saline wet-mount micros-
copy and by the qPCR assay using DNA extracted by the
QIAamp method (Table 1). For confirmation, three of the
PCR products (two from assemblage B and one from assem-
blage A) were sequenced and revealed complete identity to the
published sequence (Fig. 1). The multiplex qPCR assay dem-
onstrated 85% (83 of 97 samples) agreement with microscopy.
The one microscopy-positive and qPCR-negative specimen
was unavailable for additional testing; however, the microsco-
py-negative and qPCR-positive specimens were retested by
ELISA, and 69% (9 of 13) were ELISA positive. Additionally,
the microscopy-negative and qPCR-positive specimens had de-
layed CT values versus the microscopy-positive and qPCR-
positive specimens (35.3 ⫾ 4.5 versus 33.0 ⫾ 3.6; P ⫽ 0.04),
further suggesting that the microscopy-negative and qPCR-
positive specimens represented low-burden true infections. Fi-
nally, all available qPCR-positive specimens were retested by
ELISA; 88% (30 of 34) were ELISA positive, and qPCR CT
values exhibited the expected inverse correlation with ELISA
optical density values (correlation coefficient ⫽ ⫺0.44; P ⫽
0.01 by Spearman correlation test [n ⫽ 30]). Accordingly, with
qPCR as the “gold standard,” microscopic examination exhib-
ited 98% specificity and only 62% sensitivity.
VOL. 43, 2005
FIG. 3. Genotype-specific multiplex qPCR amplification for G. lamblia assemblages A and B. Parasite-free stool was spiked with 2,000
assemblage A trophozoites/reaction (A), 2,000 assemblage B trophozoites/reaction (B), or both (C). Multiplex qPCR was performed using the
HEX-labeled assemblage A-specific Scorpion probe and the FAM-labeled assemblage B-specific Scorpion probe. No FAM fluorescence was
detectable in the reaction represented by panel A, and no HEX fluorescence was detectable in the reaction represented by panel B.
DISCUSSION
This work details the development of a novel real-time PCR
assay that genotypes Giardia infections from fecal specimens in
a single closed-tube reaction. The test is specific and sensitive
to well below the estimated parasite excretion rate in humans
of 150 to 20,000 cysts/gram of stool (21). The assay can test 96
samples in 2 h and thus provides the capacity to characterize
the Giardia genotype for large numbers of asymptomatic and
symptomatic individuals from various geographic locations.
Such epidemiologic data are presently limited but may ulti-
mately enhance the ability to prognosticate an individual’s
course of infection and investigate source outbreaks.
We utilized Scorpion probes (30) instead of hydrolysis
probes, hybridization probes, molecular beacons, or SYBR
Green I. Although SYBR Green I was used for assay devel-
opment (data not shown), we felt that the nonspecific fluores-
cence of an intercalating dye would be unacceptable given the
complexity of fecally extracted DNA and our desire to multi-
plex. A published hydrolysis probe-based assay (13) was tried
during early development; however, this TaqMan probe did
not allow discrimination of genotypes and required a longer
amplicon, which decreased amplification sensitivity (data not
shown). Furthermore, in practice we found that genotype-spe-
cific design of TaqMan probes, hybridization probes, or mo-
lecular beacons was complicated by the high G-C content
(⬃75%) of the Giardia 18S rRNA gene, its frequent runs of
guanines, and the few nucleotide polymorphisms between as-
TABLE 1. Comparison of multiplex qPCR and microscopy for
diagnosis of Giardia infection
No. of positive qPCR results (CT ⫾ SD)
Microscopy No. of negative
result Assemblage Assemblage qPCR results
Total
A B
Positive 2 20 22 (33.0 ⫾ 3.6)a 1 qPCR-positive specimens were true infections given their high
Negative 1 12 13 (35.3 ⫾ 4.5) 61
a
P ⫽ 0.04.
GENOTYPE-SPECIFIC qPCR FOR GIARDIA LAMBLIA 1259
semblages. Moreover, these bi- or trimolecular reactions in-
crease the possibility of primer-probe dimerization or nonspe-
cific binding to DNA template and impose temperature
constraints on the kinetics of hybridization, all of which would
be magnified upon multiplexing.
Scorpion probes are unimolecular and yet can maintain a
dual layer of specificity when both primer and probe are ge-
notype specific. The primers we chose were specific for assem-
blage A and B isolates, including subgroups A-I and -II and
B-III and -IV, according to available sequence. The primer
segment of ScA contained a 3-bp mismatch to assemblage B
DNA, and the probe portion took advantage of an additional
2-bp assemblage A-specific change. This dual layer of specific-
ity ensured that falsely primed products would not lead to
fluorescence detection. Sufficient specificity and sensitivity was
achieved with an ScB Scorpion probe, with only a 3-bp mis-
match at the primer 3⬘ end, so a B-specific sequence was not
required in the probe region. An additional layer of specificity
to the PCR was added by utilization of a sequence-specific
oligonucleotide to capture our DNA of interest. This DNA-
capture technique (23) increased sensitivity at least 16-fold
versus the results seen with the widely used commercial
QIAamp stool kit, presumably due to increased target DNA
yield. We used a proprietary lysis buffer for this method; how-
ever, others have performed the same technique with conven-
tional reagents (15). On the same note, the multiplex Scorpion
PCR can be rapidly cooled to 30°C for endpoint analysis on a
fluorescent plate reader, obviating the expense of a real-time
PCR machine. These are important concerns, as the assay
should be as inexpensive as possible for the regions of the
world where Giardia is highly endemic.
The test performed favorably with human diarrheal speci-
mens and detected 60% more infections than microscopy
alone. While there is no perfect gold standard test for enteric
infections, we suspect that these microscopy-negative and
rate of ELISA positivity and relatively delayed CT. The spec-
imens were from a cohort of children with diarrhea in Mirpur,
1260 NG ET AL.
Bangladesh, and most (91%) were assemblage B infections.
This majority is similar to the 70 to 80% rates of assemblage B
infection observed in Australia (22) and Canada (9) but con-
trasts with reports from Mexico (6), India (20), and China (31),
which have indicated equal or higher assemblage A infection
rates. It is important to emphasize that the reported studies
were small and uncontrolled and that some used cultivated
trophozoites as opposed to direct fecal testing. As such, we will
await larger studies, and this is the rationale for developing this
multiplex assay.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant AI-056872
and AI-043596 from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, the Carilion Biomedical Insti-
tute, and the Commonwealth Technology Research Fund.
We thank Steven Powell for guidance, Tony Schuber for provision of
Exact Buffer A (Exact Sciences Corporation, Marlborough, Mass.),
and William A. Petri, Jr., for helpful discussions.
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