Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.
ABSTRACT Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.
- SourceAvailable from: Chihiro Sugimoto[show abstract] [hide abstract]
ABSTRACT: While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.Journal of Clinical Microbiology 01/2004; 41(12):5517-24. · 4.07 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1, 272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to both T. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.Journal of Clinical Microbiology 03/2000; 38(2):737-44. · 4.07 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.Nucleic Acids Research 07/2000; 28(12):E63. · 8.28 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2009, p. 168–174
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 1
Loop-Mediated Isothermal Amplification Method for Differentiation
and Rapid Detection of Taenia Species?
Agathe Nkouawa,1,3† Yasuhito Sako,1†* Minoru Nakao,1Kazuhiro Nakaya,2and Akira Ito1†
Department of Parasitology1and Animal Laboratory for Medical Research,2Asahikawa Medical College,
Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510 Hokkaido, Japan, and Medical Research Center,
Institute of Medical Research and Medicinal Plants Studies (IMPM), Ministry of
Scientific Research and Innovation, Yaounde ´, Cameroon3
Received 13 August 2008/Returned for modification 14 October 2008/Accepted 6 November 2008
Rapid detection and differentiation of Taenia species are required for the control and prevention of
taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and
specificity of the conventional diagnosis based on microscopical examination, molecular tools are more
reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-
mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia
species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes.
LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer
sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction
enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia
saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite
DNA samples extracted from proglottids (n ? 100) and cysticerci (n ? 68). LAMP could detect one copy
of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar
to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal
samples (n ? 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP
assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and
cysticercosis in countries where these diseases are endemic.
Cestode parasites Taenia solium, Taenia saginata, and Taenia
asiatica are the causative agents of taeniasis. Although taeniasis is
relatively innocuous, cysticercosis caused by T. solium larvae is
one of the most severe diseases in humans and remains a com-
plicated health problem in many areas around the world, espe-
cially in developing countries (4, 15). Therefore, differentiation of
Taenia species becomes significant for epidemiological studies
and for control of these diseases. Furthermore, it is expected that
there are much wider areas in the Asia-Pacific region where the
three taeniid species occur sympatrically (3, 5, 23, 24). Diagnosis
is mainly performed by microscopic observation of eggs in feces
and/or by comparative morphology of proglottids or scolices, but
these methods lack both sensitivity and specificity. In order
to overcome the lower sensitivity of microscopic diagnosis,
various immunological or molecular approaches, including
coproantigen and copro-DNA detection methods, have been
developed (2, 6, 12, 18, 25). The coproantigen detection
method has been shown to be more sensitive than the mi-
croscopy method, but it cannot differentiate between Taenia
species because of it is genus specific, not species specific
(2). By contrast, various copro-DNA detection methods us-
ing PCR have been developed for sensitive differential de-
tection of taeniid cestodes (6, 12, 18, 25). Although these
techniques provide sensitive and reliable diagnostic results,
it is not easy to exploit in the laboratories of developing
countries where these diseases are endemic, because PCR
requires sophisticated equipment, such as a thermal cycler.
Furthermore, Taq DNA polymerase is often inactivated by
inhibitors present in biological samples, which sometimes
cause problems for sensitivity and reproducibility (1, 13).
Recently, a novel nucleic acid amplification method termed
loop-mediated isothermal amplification (LAMP) has been de-
veloped (17). LAMP employs a DNA polymerase with strand
displacement activity and four primers that recognize six se-
quences on the target DNA. This method amplifies DNA with
high specificity, sensitivity, and rapidity under isothermal con-
ditions. Since LAMP is done under isothermal conditions (60
to 65°C), simple incubators, such as a water bath or a block
heater, are sufficient for DNA amplification (17). Moreover, a
large amount of white precipitate of magnesium pyrophos-
phate is produced as a byproduct, which enables the visual
judgment of amplification by a naked eye (14). Hence, LAMP
is a highly sensitive and specific DNA amplification tool suit-
able for the rapid diagnosis of infectious diseases, including
parasitic diseases (9, 20, 22), in a well-equipped laboratory
and/or small-scale clinical laboratories and is expected to be
highly useful and feasible in the field.
In the present study, we developed and evaluated the
LAMP assay with a cathepsin L-like cysteine peptidase (clp)
gene of nuclear DNA and a cytochrome c oxidase subunit 1
(cox1) gene of mitochondrial DNA for differentiation be-
tween and rapid diagnosis of infection with Taenia species.
* Corresponding author. Mailing address: Department of Parasitol-
ogy, Asahikawa Medical College, Midorigaoka Higashi 2-1-1-1, Asa-
hikawa 078-8510, Hokkaido, Japan. Phone: 81-166-68-2422. Fax: 81-
166-68-2429. E-mail: email@example.com.
† These authors contributed equally to this work.
?Published ahead of print on 12 November 2008.
MATERIALS AND METHODS
Parasite materials. Cysticerci of T. solium, T. saginata, or T. asiatica were
obtained from nonobese diabetic/severe combined immunodeficiency (NOD/shi-
scid) mice (7, 16) infected by intraperitoneal cavity injection with oncospheres
prepared from gravid proglottids of each parasite isolated in Thailand. Genomic
DNA was extracted from one cysticercus by using a DNeasy tissue kit (Qiagen,
DNA samples. For evaluation of the LAMP assay, a total of 168 DNA samples
extracted from proglottids (n ? 100) and cysticerci (n ? 68), including 47 T.
solium samples, 78 T. saginata samples, and 43 T. asiatica samples, were exam-
ined (Table 1). We used the stored DNA samples previously analyzed by mul-
tiplex PCR (25).
Cloning and sequencing of clp genes. The clp gene of each Taenia parasite was
cloned by PCR with forward primer 5?-ACATTTTCGTTTCGATCGGTCAT
G-3? and reverse primer 5?-TGAACACATGGTTTAAACGTATGG-3?. Prim-
ers used were designed from conserved nucleotide sequences between Echino-
coccus multilocularis (21) and T. solium (10) clp genes to amplify almost the
entire gene region. PCR was carried out using high-fidelity polymerase Prime-
Star (Takara, Kyoto, Japan) in a final volume of 25 ?l reaction mixture contain-
ing 0.2 ?M of each primer, 200 ?M each of deoxynucleoside triphosphate
(dNTP), 0.625 units of PrimeStar DNA polymerase, and genomic DNA, as
described above. Amplification was performed with 35 cycles of 94°C for 30 s,
60°C for 15 s, and 72°C for 4 min, followed by a final extension at 72°C for 10 min.
A single band of approximately 3 kbp was excised from agarose gels by using a
NucleoSpin ExTract kit (Macherey-Nagel, Du ¨ren, Germany) according to the
instruction manual and was cloned into a pGEM-T vector (Promega, Madison,
WI) after the addition of adenine to the ends of the PCR products. The plasmid
clone was sequenced on an ABI Prism 310 sequencer (Applied Biosystems,
Foster City, CA) with BigDye Terminator version 1.1 (AB Applied Biosystems)
and was used as a standard plasmid for determining the specificity and sensitivity
Preparation of standard plasmids for cox1 genes. The cox1 gene of each
Taenia parasite was amplified by PCR using forward primer 5?-ATGAATGTC
AAATATTTGT-TAAGTT-3? and reverse primer 5?-CTAAAAGACCATTTC
ACACGCGAAT-3? for T. solium, forward primer 5?-ATGAGTGTTAAATTT
TTATTAAGTT-3? and reverse primer 5?-TTAAACTAAAAAACCACGGGC
AGGC-3? for T. saginata, and forward primer 5?-ATGAGTGTTAAATTTTTA
TTAAGTT-3? and reverse primer 5?-TTAAACTAAAAAACCACGAGCAAA
C-3? for T. asiatica. PCR was carried out as described above, except the
annealing temperature was at 58°C and the elongation time was 90 s. The
amplified products of each Taenia species were cloned into a pGEM-T vector
and used as a standard plasmid after being confirmed by sequencing.
LAMP primers. LAMP primers were designed using PrimerExplorer V4 soft-
ware (http://primerexplorer.jp/). The following four oligonucleotide primers
were specifically designed to amplify six distinct regions on the target gene:
forward inner primer (FIP), backward inner primer (BIP), and two outer prim-
ers, F3 and B3 (Table 2). FIP consists of the sense sequence of F2 at the 3? end
and the F1c region at the 5? end that is complementary to the F1 region. BIP
consists of a B2 region at the 3? end that is complementary to the B2c region and
the same sequence as the B1c region at the 5? end (Fig. 1A and 2A).
LAMP reaction. LAMP was carried out in a 25-?l volume of reaction mixture
containing 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3, an 8-U large
fragment of Bst DNA polymerase (New England Biolabs, Beverly, MA), 20 mM
Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Tween
20, 0.8 M betaine (Sigma, St. Louis, MO), and 1.4 mM each of dNTP and the
template DNA. The reaction mixture was incubated for 60 min at 63°C for the
clp gene and at 60°C for the cox1 gene and heated at 80°C for 5 min. The LAMP
products from primer set Tsag-clp were digested with the HinfI restriction
enzyme (New England Biolabs) for 2 h at 37°C. The LAMP products and
restriction enzyme-digested products were electrophoresed on a 2.0% agarose
gel and detected by staining with ethidium bromide.
Sensitivity analyses. The sensitivities of the LAMP and PCR methods were
assessed using a standard plasmid diluted from 104copies/reaction to 1. LAMP
was performed as previously mentioned. PCR was carried out by using F3 and B3
primers of each LAMP primer set. PCRs were performed in a 25-?l volume of
reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 0.001% gelatin, 0.2 ?M of each primer, 0.2 mM each of dNTP, diluted
standard plasmid, and 0.5 units of Taq DNA polymerase (ExTaq; Takara), and
cycling conditions were 30 s at 94°C (first cycle, 2 min at 94°C), 30 s at 60°C (clp
gene) and 58°C (cox1 gene), and 30 s at 72°C for 30 cycles. The PCR products
were electrophoresed on a 2.0% agarose gel and detected by staining with
DNA extraction from fecal samples. In order to evaluate the detection limit of
taeniid DNA in fecal samples, 1 gram of feces from a noninfected volunteer was
mixed with 5, 10, 20, 30, 40, or 50 T. asiatica or T. saginata eggs, and DNA was
extracted by using a QIAamp DNA stool mini kit (Qiagen) after egg disruption
treatment with glass beads (19). In addition, taeniid egg-positive or -negative
fecal samples obtained in Indonesia and Thailand were used to assess LAMP,
and DNA samples were extracted by the same procedure. LAMP was performed
as previously described except that the reaction was performed for 90 min.
Nucleotide sequence accession numbers. The nucleotide sequence data of the
clp genes cloned in this study are available in the GenBank, EMBL, and DDBJ
databases under accession numbers AB441815 (T. solium), AB441816 (T. sagi-
nata), and AB441817 (T. asiatica).
RESULTS AND DISCUSSION
The standard plasmid of each gene was constructed to facil-
itate initial evaluation and optimization of LAMP. The LAMP
products are detected as a ladder of multiple bands on the gel
due to the formation of a mixture of stem-loop DNAs with
various stem lengths and cauliflower-like structures, with mul-
tiple loops formed by annealing between alternately inverted
repeats of the target sequence in the same strand (17). Appli-
cation of the LAMP assay with each specific primer set to each
TABLE 1. Taenia species DNA samples used in this study
(no. of samples)
(no. of samples analyzed)
T. saginata (78)Cambodia
T. asiatica (43)China Cysticercus (1)a
T. solium (47)China Cysticercus (2)
aThe cysticercus was developed in NOD/shi-scid mice.
VOL. 47, 2009LAMP METHOD FOR TAENIA SPECIES DIFFERENTIATION 169
Taenia cox1 gene resulted in successful amplification of the
target gene from the respective parasite genomic DNA (Fig.
1B). On the other hand, because of high nucleotide sequence
similarity between the clp genes of T. saginata and T. asiatica,
we could not design a specific primer set to differentiate be-
tween these two Taenia species but could design specific
primer sets Tsol-clp and Tsag-clp to differentiate T. solium
from T. saginata and T. asiatica and differentiate T. saginata/T.
asiatica from T. solium, respectively. However, the recognition
site GAGTC for the restriction enzyme HinfI in the amplified
region of the T. asiatica clp gene (Fig. 2A) enables us to
differentiate T. asiatica from T. saginata. In this case, restric-
tion enzyme digestion of the LAMP products from T. asiatica
genomic DNA with primer set Tsag-clp produces three bands
with the predicted sizes of 179, 217, and 255 bp, because,
unlike the PCR products, the LAMP products are character-
istic structures with inverted repeats of the target sequence. In
fact, three bands that agreed with the predicted size in the
restriction enzyme digestion of the LAMP products from T.
asiatica genomic DNA with primer set Tsag-clp were detected
(Fig. 2B, lane 6).
Analytical specificity of the LAMP assays. In order to eval-
uate the specificity of the LAMP assays, the known parasite
DNA samples prepared from proglottids and cysticerci were
examined. Because LAMP requires four primers that rec-
ognize six different sequences on a target sequence, the
target sequence specificity of the LAMP reaction appears to
be high. Indeed, LAMP with primer sets to the cox1 or clp
gene specifically amplified each respective target gene, with
a species-specific detection. The results obtained by LAMP
with cox1 primer sets were consistent with those obtained by
multiplex PCR with the cox1 gene, whereas two DNA sam-
ples extracted from proglottids, which were identified as
being T. saginata by both LAMP and multiplex PCR with
cox1 primer sets, were detected as T. asiatica by LAMP with
clp primer sets (Table 3). The size of bands produced by
restriction enzyme digestion indicated that these two LAMP
products were specifically derived from the T. asiatica clp
gene (data not shown). After cloning of the clp gene from
these two DNA samples by PCR with primer set Tsag-clp F3
and B3 and sequencing, we found a single nucleotide sub-
stitution, T to C, as indicated in Fig. 2A, which leads to the
appearance of the HinfI recognition site in the region be-
tween B1c and B2 and is identical to the nucleotide substi-
tution in T. asiatica. Thus, the nucleotide substitution in the
clp gene of these two T. saginata samples may lead to the
different result from the diagnosis based on the cox1 gene.
Alternatively, the possibility that these two DNA samples
were obtained from a hybrid parasite having T. asiatica
nuclear DNA and T. saginata mitochondrial DNA could not
be ruled out, because samples of these parasite materials
were collected from areas where both parasites exist sym-
patrically in China and Thailand, respectively, and these
materials were identified by only mitochondrial DNA (3, 8).
Exactly two tapeworms obtained in Thailand where three
human Taenia species were confirmed to be sympatrically
occurring (3) are concluded to be the hybrids of T. saginata
and T. asiatica (M. Okamoto, M. Nakao, M. T. Anan-
taphruti, J. Waikagul, and A. Ito, unpublished data). More-
detailed characterizations of these two DNA samples must
be performed using another nuclear DNA marker, in addi-
tion to analyses of longer nucleotide sequences of the clp
genes. These results demonstrated that the LAMP methods
with each primer set designed in this study can specifically
TABLE 2. LAMP primer sets
SpeciesPrimer set Primer typea
Sequence (5? to 3?) Target
T. saginataTsag-cox1 F3
T. asiatica Tasi-cox1 F3
T. solium Tsol-cox1F3
T. saginataTsag-clpF3 GGAAGTCAAAAATCAGGTGAGclp gene
T. asiatica B3
T. soliumTsol-clp F3
aFIP consists of the sense sequence of F2 at the 3? end and the F1c region at the 5? end that is complementary to the F1 region. BIP consists of a B2 region at the
3? end that is complementary to the B2c region and the same sequence as the B1c region at the 5? end.
170 NKOUAWA ET AL.J. CLIN. MICROBIOL.
amplify the target gene and are applicable to a differential
diagnosis of infections with Taenia species.
Analytical sensitivity of the LAMP assay. Tenfold serial
dilution of each standard plasmid was used to determine the
lower detection limits for LAMP and PCR, and the sensitivities
of the two methods were compared. In this study, F3 and B3
primers of each LAMP primer set were utilized for PCR.
Figure 3A shows results of LAMP and PCR with primer set
Tsol-clp. Both LAMP and PCR detected up to one copy of
target gene/reaction, which indicated no difference in sensitiv-
ity between the two methods. LAMP and PCR with other
primer sets provided the same results as LAMP with primer set
Tsol-clp (data not shown).
Next, the detection limit of taeniid eggs in feces was
evaluated using T. asiatica and T. saginata eggs (Fig. 3B). At
least five eggs per gram of feces was sufficient for taeniid
eggs to be detected by LAMP with primer sets Tasi-cox1 and
Tsag-cox1, whereas more than 10 eggs/g of feces was needed
to be detected by LAMP with primer set Tsag-clp (data not
shown). The differences between the cox1 gene and the clp
gene in the detection sensitivity may be responsible for the
number of copies of each target gene within the samples,
since a large number of mitochondrial DNA exists in a cell,
one feature to be selected as a target DNA for detection.
Several detection methods for Taenia species in feces based
on PCR techniques have been reported. The multiplex PCR
method with mitochondrial DNA (25), the PCR-restriction
fragment length polymorphism method with mitochondrial
DNA (18), and the nested-PCR method with the Tso31 gene
encoding the T. solium oncosphere-specific protein (12)
have been reported to show detection limits of 5 eggs/g of
feces, 17 eggs/g of feces, and 40 eggs/g of feces, respectively.
It seems that more eggs are needed for detection when the
nuclear gene is chosen as a diagnostic gene marker, al-
though the amplification efficiency of each PCR method
varies. In this study, T. solium eggs were not available, but a
similar sensitivity might be expected because all LAMP
primer sets could amplify one copy of the target gene when
using pure plasmid DNA samples.
Differential detection of Taenia species in fecal samples.
Furthermore, in order to assess the LAMP method for the
detection of copro-DNA, we investigated fecal samples that
FIG. 1. Nucleotide sequence alignment of the target region of cox1 genes (A) and LAMP results (B). (A) The locations of the primer
recognition sites are indicated by arrows. (B) The LAMP products were run on a 2% agarose gel. Lane M, 100-bp DNA ladder marker (Promega);
lane 1, negative control; lane 2, T. saginata genomic DNA; lane 3, T. asiatica genomic DNA; lane 4, T. solium genomic DNA; Tsag-cox1, results
of LAMP with primer set Tsag-cox1; Tasi-cox1, results of LAMP with primer set Tasi-cox1; Tsol-cox1, results of LAMP with primer set Tsol-cox1.
VOL. 47, 2009LAMP METHOD FOR TAENIA SPECIES DIFFERENTIATION 171
were positive (n ? 6) or negative (n ? 10) for taeniid eggs
by microscopy and collected in Indonesia and Thailand (Fig.
4). Out of six taeniid egg-positive fecal samples, three spec-
imens from Indonesia tested positive with primer sets Tsag-
cox1 (Fig. 4A, lanes 15 to 17) and Tsag-clp (data not shown),
and the remaining specimens from Thailand were positive
with primer sets Tasi-cox1 (Fig. 4B, lanes 12 to 14) and
Tsag-clp (data not shown). No differences in detection be-
FIG. 2. Nucleotide sequence alignment of the target region of clp genes (A) and LAMP results (B). (A) The locations of the primer recognition sites
are indicated by arrows and the restriction enzyme HinfI recognition site in the LAMP products of T. asiatica clp gene is boxed. The single nucleotide
substitution, C to T, in the T. saginata sequence is indicated by an arrowhead. (B) The LAMP products and HinfI digestion products were run on a 2%
of LAMP products from T. asiatica genomic DNA with primer set Tsag-clp; Tsag-clp, results of LAMP with primer set Tsag-clp; D, results of HinfI digestion;
Tsol-clp, results of LAMP with primer set Tsol-clp. The DNA fragments generated after digestion with HinfI are indicated by arrowheads.
TABLE 3. Analytical specificity of the LAMP assaysa
(no. of samples)
No. of samples (%) detected by LAMP
with indicated primer set
T. saginata (78)
T. asiatica (43)
T. solium (47)
aTaenia species were confirmed by multiplex PCR with cox1 genes (25).
bTwo samples were detected as T. asiatica by LAMP.
FIG. 3. (A) Comparison of detection sensitivities of LAMP and
PCR with Tsol-clp primer set. The standard plasmids were serially
diluted from 104copies per reaction to 1 copy per reaction and am-
plified by LAMP (upper panel) and PCR (lower panel). The F3 and B3
primers of the Tsol-clp primer set were used in the PCR. The LAMP
reactions were carried out for 60 min. Lane M, 100-bp DNA ladder
marker (Promega); lanes 1 to 5 represent 104, 103, 102, 10, and 1
copy(ies)/reaction, respectively; lane 6, negative control. (B) Detection
limits of target genes by LAMP with Tasi-cox1 primer set against DNA
samples prepared from feces containing various numbers of T. asiatica
eggs. The LAMP reactions were carried out for 90 min. Lane M,
100-bp DNA ladder marker; lane 1, negative control; lane 2, T. asiatica
genomic DNA as a positive control; lanes 3 to 8 represent 5, 10, 20, 30,
40, and 50 T. asiatica eggs/g of feces, respectively.
172 NKOUAWA ET AL.J. CLIN. MICROBIOL.