497944

research-article2013
VDIXXX10.1177/1040638713497944Real-time PCR for the differential detection of ParagonimusTantrawatpan et al.

Article

Journal of Veterinary Diagnostic Investigation

Application of a real-time fluorescence 25(5) 620­–626

© 2013 The Author(s)
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DOI: 10.1177/1040638713497944
jvdi.sagepub.com
reaction assay with melting curve analysis for
the detection of Paragonimus heterotremus
eggs in the feces of experimentally infected cats

Chairat Tantrawatpan, Pewpan M. Intapan,1 Tongjit Thanchomnang,

Oranuch Sanpool, Penchom Janwan, Viraphong Lulitanond, Witthaya Anamnart,
Wanchai Maleewong

Abstract. Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis
in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase
chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs
in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was
3 × 102 copies of the positive control plasmid and 10-3 ng of P. heterotremus genomic DNA. The assay system could detect
10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other
organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all
fecal samples from 17 P. heterotremus–infected cats and showed a negative relationship (r = –0.852, P < 0.001) between the
number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus,
P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from
the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can
be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas.

Key words: Egg detection; feces; parasite; real-time fluorescence resonance energy transfer polymerase chain reaction;
second internal transcribed spacer.

Paragonimiasis is an important food-borne parasitic zoonosis The standard diagnosis of paragonimiasis is based on the
caused by a lung fluke belonging to the genus Paragoni- demonstration of the presence of the eggs of Paragonimus
mus.2,10 It is estimated that more than 20 million people are spp. in the sputum (by alkaline decontamination and centri-
infected worldwide,21 and it has been calculated that fuge sedimentation technique) and/or feces (by formalin–ether
293 million people are at risk.9 Human beings and other concentration technique).1,22 However, species identification
mammals are infected by ingesting raw crustaceans contain-
ing metacercariae22 and usually present with signs and symp- From the Research and Diagnostic Center for Emerging Infectious
toms in the lower respiratory tract (i.e., cough and Diseases (Tantrawatpan, Intapan, Thanchomnang, Sanpool, Janwan,
hemoptysis).2 Paragonimus westermani is the most widely Lulitanond, Maleewong), the Departments of Parasitology (Intapan,
distributed species in Asia and the most important human Sanpool, Janwan, Maleewong) and Microbiology (Lulitanond), Faculty
of Medicine, Khon Kaen University, Khon Kaen, Thailand; the Division
pathogen in China, Korea, and Japan.2,10 In Southeast Asia, of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine,
however, P. heterotremus is the most important pathogen, Thammasat University, Rangsit Campus, Pathum Thani, Thailand
and confirmed human cases have been found.2,10 To date, at (Tantrawatpan); the Faculty of Medicine, Mahasarakham University,
least 7 lung fluke species have been documented in Thailand: Mahasarakham, Thailand (Thanchomnang); and the Department of
Paragonimus heterotremus, P. siamensis, P. westermani, P. Medical Technology, School of Allied Health Sciences and Public Health,
Walailak University, Thasala, Thailand (Anamnart).
bangkokensis, P. macrorchis, P. harinasutai, and P. pseudo- 1
Corresponding Author: Pewpan M. Intapan, Department of
heterotremus.3,19,20 However, only P. heterotremus and Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen,
P. pseudoheterotremus are proven human pathogens.6 Thailand. pewpan@kku.ac.th
 Real-time PCR for the differential detection of Paragonimus 621

of parasite eggs in the sputum and/or feces by microscopic The PhITS2_F and PhITS2_R primers targeting the
methods is tedious and requires well-trained personnel. nuclear ribosomal second internal transcribed spacer (ITS2)
Molecular methods based on the polymerase chain reaction region of Paragonimus spp. as well as the PhITS2_LC 640
(PCR) have been used for the detection and/or species dis- and PhITS2_FL 530 probes were designed and selected based
crimination of Paragonimus spp. (e.g., conventional PCR,7 on partial consensus sequences of all 6 Paragonimus spp.
random amplified polymorphic DNA-PCR,5 and multiplex using commercial software.d The genus-specific forward
PCR17). As an effective molecular method, a real-time fluo- primer PhITS2_F (5’-TGGGGTGCCAGATCTATGG-3’)
rescence resonance energy transfer PCR (real-time FRET and the reverse primer PhITS2_R (5’-GGGTACTCACGTC
PCR) has been developed as a diagnostic tool for the detec- TAATCCGAG-3’)e were expected to amplify a 231-bp frag-
tion and species differentiation of various parasites (e.g., the ment of the ITS2 sequence of P. heterotremus. A pair of
detection of Opisthorchis viverrini,16 Clonorchis sinensis,16 adjacent oligoprobes, the first labeled at the 5’-end (PhITS2_
and Schistosoma japonicum18). LC 640; 5’ Red 640-TGGTCTGTGTCCGATGCTGACC
In the current study, a rapid and high throughput real-time TATATATGTGCC-Phosphate 3’) and the second at the
FRET PCR assay combined with melting curve analysis was 3’-end (PhITS2_FL; 5’-GTTCCGCTGTCCCGTCATCATC
developed for the alternative detection of P. heterotremus TATGGTTGAAGTTGCG-Fluo 530-3’)f were used to detect
eggs in the feces of experimentally infected cats. The appli- the Paragonimus-specific ITS2 product. For control plasmid
cability of the method for differentiation of P. heterotremus preparations, the ITS2 PCR products of 6 Paragonimus spp.
from 5 other Paragonimus spp. reported in Thailand was were obtained by conventional PCR using the PhITS2_F and
also tested. The detection limit and analytical specificity of PhITS2_R primers. This PCR was performed using 1 µl of
the assay were evaluated. template DNA, 0.2 µM of each primer, 2.5 µl of 10× PCR
Fecal samples were collected from 17 cats experimentally buffer (1.5 mM MgSO4 and 0.2 mM of each deoxyribonucle-
infected with 30 P. heterotremus metacercariae per cat and otide triphosphate), and 0.5 U Taq DNA polymerase. The
from 3 uninfected cats. The fecal samples and P. heterotre- amplification procedure was 95°C for 5 min; 35 cycles of
mus eggs used in the present study were the samples remain- 95°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec; and
ing from a previous study11 and were kept frozen at –80°C 72°C for 7 min. A 231-bp PCR product for P. heterotremus,
until use. All frozen fecal samples were thawed, and the con- a 228-bp PCR product for P. macrorchis, and 233-bp PCR
dition of the preserved Paragonimus spp. eggs was reexam- products for P. westermani, P. siamensis, P. harinasutai, and
ined using a simple wet-mount smear method. The number P. bangkokensis were cloned into a commercial vector,g
of eggs was counted using the modified formalin–ether sedi- according to manufacturer’s instructions. The recombinant
mentation technique1 for P. heterotremus eggs (range of plasmids were transformed and propagated in Escherichia
eggs per gram of feces [EPG] = 50–2,950). For sensitivity coli JM109, and these plasmids were used as the control plas-
testing, feces from uninfected cats were artificially mixed mids. The nucleotide sequences of the inserted genes within
with various numbers of P. heterotremus eggs prior to DNA plasmid were sequenced in both directions before using as the
extraction. Frozen cat feces (100 mg each) were homoge- positive control of each species. The inserted sequences of
nized with disposable polypropylene pestles,a and genomic P. heterotremus, P. macrorchis, P. westermani, P. siamensis,
DNA (gDNA) was individually extracted using a commer- P. bangkokensis, and P. harinasutai control plasmids were
cial kit,b according to manufacturer’s protocol. Fecal DNA found to be identical with those in GenBank, which had been
samples were eluted in 100 μl of distilled water, of which 5 used to design the primers in the current study (accession nos.
μl was used for the real-time FRET PCR. AF159603, AF159608, AB354214, AB354222, AB248091,
The metacercariae of P. heterotremus, P. westermani, and AB354219, respectively).
P. macrorchis, P. siamensis, P. harinasutai, and P. bangko- The real-time PCR was performed using a commercial
kensis were harvested from naturally infected fresh water system.h Each glass capillary contained 1× commercial DNA
crabs. The species of each Paragonimus metacercaria was mastermix,i 2 mM MgCl2, 0.3 μM PhITS2_F primer, 0.6 μM
determined based on microscopic morphology before DNA PhITS2_R primer, 0.2 µM each of the PhITS2_FL 530 and
extraction. Metacercariae of each Paragonimus spp. were PhITS2_LC 640 probes, and each extracted DNA sample in
classified and identified by shape and size of cyst, cyst wall a 20-μl total volume. A single initial denaturation step of 10
thickness, and larval body feature (i.e., shape, arrangement, min at 95°C was followed by 45 cycles of repeated denatur-
color, and ventral sucker to oral sucker ratio).3,19,20 One meta- ation (10 sec at 95°C), annealing (30 sec at 50°C), and exten-
cercaria each of P. heterotremus, P. westermani, P. macror- sion (15 sec at 72°C). The temperature transition rate was
chis, P. siamensis, P. harinasutai, and P. bangkokensis were 20°C/sec. The amplification program was followed by a
separately homogenized with a disposable polypropylene melting curve program of 95°C for 10 sec, 49°C for 20 sec,
pestle, and DNA was extracted using a commercial kit,c and then 49°C to 75°C at a rate of 0.2°C/sec with continuous
according to the manufacturer’s protocol. Genomic DNA monitoring of fluorescence. The color compensation for fluo-
from each Paragonimus spp. was eluted in 50 μl of distilled rescence signals was performed according to the manufacturer’s
water, of which 5 μl was used for the real-time FRET PCR. instructions.j Each sample was analyzed in duplicate. Sterile
622
Tantrawatpan et al.

distilled water and a P. heterotremus positive control plas- extracted from the 3 samples of uninfected cat feces (Fig. 2A).
mid DNA (3 × 107 copies/reaction) were used as negative Although the gDNA from several parasites (e.g., S. japoni-
and positive controls, respectively, for the evaluation of the cum, C. philippinensis, S. stercoralis, and lecithodendriid
within-determination (intra-assay) and between-determination flukes) were amplified as 231-bp bands (Supplementary fig-
(interassay) variations in the melting temperature (Tm). The ure), no specific fluorescence signal was detected by the
amplicons of the positive samples were sequenced with melting peak analysis. The coefficients of the variation of
M13 universal primers in both directions by the Sanger this assay showed that all the tests were within the statisti-
method15 and the sequence analyzed using commercial cally acceptable range of day-to-day variation (data not
software.k shown).
To verify the analytical sensitivity of the detection Real-time FRET PCR combined with melting curve analy-
method, 5 μl of P. heterotremus control plasmid DNA sam- sis of the amplicon products was applied to detect DNA
ples containing between 3 × 107 and 3 copies, and samples of extracted from P. heterotremus eggs in infected cat feces. The
gDNA (1 μl) containing 10–10-5 ng of DNA extracted from a assay yielded satisfactory results, with all 17 stool samples
P. heterotremus adult were tested. To determine the sensitiv- that were positive for P. heterotremus eggs by the simple
ity of the detection of eggs in feces, 5-μl aliquots of the DNA smear method being positive by PCR. The fecal samples from
extracted from 100 mg of uninfected cat feces spiked with 1, 3 uninfected cats were negative. The results of the melting
2, 4, 8, and 16 P. heterotremus eggs (equivalent to 10, 20, 40, curve analyses for P. heterotremus DNA are shown in Figure
80, and 160 EPG, respectively) were also tested. 2B. The range, mean ± SD, and median of the Tm values for
For the evaluation of the analytical specificity, gDNA P. heterotremus DNA in the fecal samples were 68.23–68.46,
(5 ng/μl) from various parasites and 1 bacterial species was 68.32 ± 0.08, and 68.30, respectively. Furthermore, a nega-
analyzed. The panel of other organisms used for the real- tive relationship (r = –0.852, P < 0.001) was observed
time FRET PCR analysis included Schistosoma mekongi, S. between the number of P. heterotremus eggs in 100 mg of cat
japonicum, Haplorchis taichui, Fasciola gigantica, O. viver- feces and the Ct indicative of the presence of P. heterotremus
rini, Echinostoma malayanum, Clonorchis sinensis, Capil- DNA. The 231-bp PCR amplicons observed for all infected
laria philippinensis, human hookworms, Strongyloides fecal samples were sequenced in both directions, and the
stercoralis, Taenia spp., Ascaris lumbricoides, Trichuris nucleotide sequences of all amplicons were identical to the
trichiura, lecithodendriid flukes, Giardia lamblia, and reported P. heterotremus ITS2 sequence (AF159603).
Mycobacterium tuberculosis. Cat and human gDNA as well When the plasmid and gDNA from P. heterotremus, P.
as DNA from 3 samples of uninfected cat feces were also westermani, P. macrorchis, P. siamensis, P. harinasutai, and
included. P. bangkokensis were used for species differentiation using
For evaluation of the detection capacity of P. heterotre- the real-time FRET PCR assay with the melting curve analy-
mus eggs in feces, 5 μl of DNA extracted from the feces of sis, all 6 Paragonimus species could be detected, and were
17 experimentally infected cats was tested. The correlation differentiated into 4 groups based on different Tm values
between the number of P. heterotremus eggs in 100 mg of (Fig. 3). Group 1 contained only P. heterotremus (Tm values
cat feces and the threshold cycle (Ct) indicative of the pres- of plasmid DNA = 68.40 and gDNA = 68.36). Group 2 con-
ence of P. heterotremus DNA was analyzed using Spearman tained P. macrorchis (Tm values of plasmid DNA = 62.04
rank correlation test.l In addition, 5 μl of plasmid DNA (107 and gDNA = 62.01). Group 3 consisted of P. siamensis (Tm
copies) and 5 μl (1 ng/μl) of gDNA from P. heterotremus, P. values of plasmid DNA = 54.85 and gDNA = 54.82) and P.
westermani, P. macrorchis, P. siamensis, P. harinasutai, and westermani (Tm values of plasmid DNA = 54.59 and
P. bangkokensis metacercariae were evaluated for species gDNA = 54.66), which had almost equal Tm values. Group
differentiation. 4 consisted of P. harinasutai (Tm values of plasmid DNA =
The detection limit of the ITS2 target DNA sequence was 53.59 and gDNA = 53.48) and P. bangkokensis (Tm values
3 × 102 copies of the positive control plasmid (Fig. 1A) and of plasmid DNA = 53.42 and gDNA = 53.39), which also
10-3 ng of P. heterotremus gDNA (Fig. 1B), when using 35 had equal Tm values.
cycles as the cutoff detection limit. The range, mean ± stan- The current study was designed to develop a real-time
dard deviation (SD), and median of the Tm values for the P. FRET PCR assay combined with melting curve analysis to
heterotremus positive control plasmid and the gDNA were detect the ITS2 DNA sequence of P. heterotremus. The ribo-
68.28–68.51, 68.43 ± 0.08, 68.45 and 68.43–68.72, 68.54 ± somal DNA ITS2 sequences have been used for the detection
0.11, 68.52, respectively. A detection limit for P. heterotre- and species identification of various Paragonimus species.2
mus eggs in feces as low as 10 EPG of control uninfected cat Using 35 cycles as the cutoff for positivity, the lowest detec-
feces was achieved (Fig. 1C). Moreover, no fluorescence tion limit for the P. heterotremus positive control plasmid
signals (Ct values all >35) were observed for purified DNA was approximately 3 × 102 copies and 10-3 ng of P. hetero-
from 15 parasites other than P. heterotremus, gDNA from M. tremus gDNA. The assay could also detect as few as 10 EPG
tuberculosis, DNA from cats and human beings, and DNA of P. heterotremus eggs mixed in uninfected cat feces and
 Real-time PCR for the differential detection of Paragonimus 623

Figure 1.  Amplification plots of fluorescence ( y-axis) versus threshold cycle (Ct; x-axis) show the detection limit of the real-time
fluorescence resonance energy transfer polymerase chain reaction assay. A, Paragonimus heterotremus plasmid DNA in 10-fold dilutions
containing 3 × 107 copies (a; Ct = 12.91), 3 × 106 copies (b; Ct = 16.50), 3 × 105 copies (c; Ct = 19.99), 3 × 104 copies (d; Ct = 23.88), 3 ×
103 copies (e; Ct = 28.12), 3 × 102 copies (f; Ct = 31.60), 30 copies (g; Ct > 35), and 3 copies (h; Ct > 35). Distilled water (i; Ct > 35) was
included as a control. B, genomic DNA from P. heterotremus at concentrations of 10 ng ( j; Ct = 21.15), 1 ng (k; Ct = 24.31), 1 × 10-1 ng (l;
Ct = 28.11), 1 × 10-2 ng (m; Ct = 32.02), 1 × 10-3 ng (n; Ct = 34.99), 1 × 10-4 ng (o; Ct > 35), and 1 × 10-5 ng (p; Ct > 35). C, DNA extracted
from a known number of P. heterotremus eggs in the 100 mg feces of uninfected cats: 16 eggs (q; Ct = 30.69), 8 eggs (r; Ct = 31.49), 4 eggs
(s; Ct = 33.26), 2 eggs (t; Ct = 34.72), 1 egg (u; Ct = 34.92), and uninfected cat feces (v; Ct > 35). Paragonimus heterotremus plasmid DNA
at 3 × 107 copies (a; Ct = 12.97) and distilled water (i; Ct > 35) were used as positive and negative controls, respectively.

also could detect P. heterotremus eggs in the feces of all was at approximately 8–10 EPG.7 Further improvement of
experimentally infected cats. Although the detection of the sensitivity of detection is needed.
P. heterotremus gDNA was 10 times less sensitive than the In the current study, no fluorescence signal was observed
conventional PCR detection reported previously,7 the detec- for DNA samples from 15 parasites other than members of
tion limit of this method is quite similar to that for the detec- the genus Paragonimus, M. tuberculosis, cats, and human
tion of P. heterotremus eggs, for which the detection limit beings, showing the high specificity of this protocol. The
624
Tantrawatpan et al.

Figure 2.  Specificity analysis of the real-time fluorescence resonance energy transfer polymerase chain reaction assay. A, melting
curves for 3 × 107 copies of Paragonimus heterotremus plasmid DNA (a; threshold cycle [Ct] = 13.01) as positive control and purified DNA
from 15 parasites other than P. heterotremus and genomic DNA from Mycobacterium tuberculosis, cats, and human beings; and DNA from
3 samples of uninfected cat feces (b). B, melting curves were also analyzed for 17 samples of feces from cat infected with P. heterotremus
(c; mean Ct ± standard deviation = 27.70 ± 1.67). No fluorescence signal was observed for the 3 samples of uninfected cat feces and distilled
water (d).

metacercariae of different species of lung flukes can be

found in the same freshwater crab species (e.g., P. bangko-
kensis and P. harinasutai metacercariae in Potamon smithia-
num4,13; P. heterotremus, P. bangkokensis, and P. harinasutai
metacercariae in Potamon lipkei14; and P. heterotremus, P.
westermani, and P. harinasutai metacercariae in the fresh-
water crab Larnaudia larnaudii in Thailand8). The species
identification of the eggs and metacercariae of these lung
flukes requires careful morphological examination by an
expert.12 The PhITS2_LC 640 and PhITS2_FL probes used
in the current study were designed to detect the partial con-
sensus sequences of the ITS2 of all 6 Paragonimus spp.
occurring in Thailand. The partial ITS2 sequences of Para-
gonimus spp. were aligned using 2 probes that varied from
the known sequence in the following manner: a 3-nucleotide
Figure 3. Representative melting curves constructed using difference for P. heterotremus (GenBank accession no.
2 fluorophore-labeled probes hybridized to the amplification AF159603); a 10-nucleotide difference for P. macrorchis
products of the second internal transcribed spacer from 107 copies (AF159608), P. westermani (AB354214), and P. siamensis
of plasmid DNA (a, c, e, g, i, k) and 5 ng of genomic DNA (gDNA; (AB354222); and a 12-nucleotide difference for P. bangko-
b, d, f, h, j, l) from 6 species of Paragonimus. The melting curves
for this experiment are shown as –(d/dT) fluorescence (640/530).
kensis (AB248091) and P. harinasutai (AB354219). The
Group 1: P. heterotremus plasmid DNA (a; melting temperature mismatches between the probes and the parasite DNA tem-
[Tm] = 68.40) and gDNA from P. heterotremus (b; Tm = 68.36); plate resulted in different Tm values. The results of the melt-
group 2: P. macrorchis plasmid DNA (c; Tm = 62.04) and gDNA ing point analysis show that the 6 Paragonimus spp.
from P. macrorchis (d; Tm = 62.01); group 3: P. siamensis plasmid examined in the present study could be divided into 4 groups
DNA (e; Tm = 54.85), gDNA from P. siamensis (f; Tm = 54.82), with different Tm values; P. heterotremus and P. macrorchis
P. westermani plasmid DNA (g; Tm = 54.59), and gDNA from P. each form a distinct group, P. siamensis and P. westermani
westermani (h; Tm = 54.66); group 4: P. harinasutai plasmid DNA
(i; Tm = 53.59), gDNA from P. harinasutai ( j; Tm = 53.48), P.
make up a group, and P. bangkokensis and P. harinasutai
bangkokensis plasmid DNA (k; Tm = 53.42), and gDNA from P. make up another group. This grouping is in agreement with
bangkokensis (l; Tm = 53.39) were included. Distilled water (m) the molecular phylogenetic tree analysis using ITS2
was used as a negative control in this study. sequences performed in a previous study, with P. siamensis
 Real-time PCR for the differential detection of Paragonimus 625

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