Differential Detection of Trichinella Papuae, T. Spiralis and T

Veterinary Parasitology 185 (2012) 210–215

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Veterinary Parasitology

jou rnal homepage: www.elsevier.com/locate/vetpar

Differential detection of Trichinella papuae, T. spiralis and

T. pseudospiralis by real-time ﬂuorescence resonance energy transfer

PCR and melting curve analysis

a,b a,c a,d

Chairat Tantrawatpan , Pewpan M. Intapan , Tongjit Thanchomnang ,

a,e c f g

Viraphong Lulitanond , Thidarut Boonmars , Zhiliang Wu , Nimit Morakote , a,c,∗

Wanchai Maleewong

Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen 40002, Thailand

Division of Cell Biology, Department of Preclinical Sciences, Thammasat University, Rangsit Campus, Pathum Thani 12121, Thailand

Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

Faculty of Medicine, Mahasarakham University, Mahasarakham 44150, Thailand

Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

Department of Parasitology, Gifu University Graduate School of Medicine, Yanagido1-1, Gifu 501-1194, Japan

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease.

Received 6 July 2011

Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and

Received in revised form

Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand.

26 September 2011

Deﬁnite diagnosis of this helminthiasis is based on a ﬁnding of the Trichinella larva (e) in a

Accepted 30 September 2011

muscle biopsy. The parasite species or genotype can be determined using molecular meth-

ods, e.g., polymerase chain reaction (PCR). This study has utilized real-time ﬂuorescence

Keywords:

resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the

Trichinella spiralis

differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were

Trichinella papuae

Trichinella pseudospiralis studied using one set of primers and ﬂuorophore-labeled hybridization probes speciﬁc for

Differentiation the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as

Detection the cut-off for positivity and using different melting temperatures (Tm), this assay detected

Real-time FRET PCR

T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean Tm ± SD val-

± ± ±

ues to be 51.79 0.06, 66.09 0.46 and 51.46 0.09, respectively. The analytical sensitivity

of the technique enabled the detection of a single Trichinella larva of each species, and the

detection limit for the target DNA sequence was 16 copies of positive control plasmid.

A test of the technique’s analytical speciﬁcity showed no ﬂuorescence signal for a panel

of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensi-

tivity and speciﬁcity of the detection of these Trichinella species, as well as the fast and

high-throughput nature of these tools, this method has application potential in differenti-

ating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of

∗

Corresponding author at: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Tel.: +66 43 348387;

fax: +66 43 202475.

E-mail address: wanch [email protected] (W. Maleewong).

C. Tantrawatpan et al. / Veterinary Parasitology 185 (2012) 210–215 211

1. Introduction T. spiralis and T. pseudospiralis, which all are indigenous to

Thailand.

Trichinellosis is a worldwide zoonotic disease caused

by the consumption of uncooked meat containing nema- 2. Materials and methods

todes of the genus Trichinella. There are approximately

10,000 human trichinellosis infections per year, and the 2.1. Larva isolates and experimental animals

mortality rate is 0.2% in the case of severe infections

(Dupouy-Camet and Murell, 2007; Gottstein et al., 2009). T. spiralis, T. pseudospiralis and T. papuae were main-

Currently, the genus Trichinella is classiﬁed into eight tained in the laboratory. T. spiralis caused of an outbreak

species and four genotypes within two clades. The encap- in the Mae Hong Son Province in 1986 (Pozio and

sulated clade consists of ﬁve species (Trichinella spiralis, Khamboonruang, 1989). T. pseudospiralis (ISS13) was

Trichinella nativa, Trichinella britovi, Trichinella murrelli and provided by the Department of Parasitology, the Gifu Uni-

Trichinella nelsoni) and four genotypes (Trichinella T6, T8, T9 versity Graduate School of Medicine, Japan. T. papuae

and T12), whereas the non-encapsulated clade consists of was derived from a patient who had worked in Malaysia

three species (Trichinella pseudospiralis, Trichinella papuae and who had a history of having eaten hunted raw

and Trichinella zimbabwensis) (Pozio and Zarlenga, 2005; wild bear in 2005 (Chotmongkol et al., 2005). This iso-

Krivokapich et al., 2008). late was later identiﬁed as T. papuae by DNA sequencing

Over 118 outbreaks of trichinellosis have been reported of both the small subunit of ribosomal RNA and the

in Thailand since 1962, and there have been approxi- cytochrome c oxidase subunit I genes in 2010 (Intapan

mately 5500 people affected and 95 deaths (Boonthanom et al., 2011). In this study, mice were orally infected

and Nawarat, 1963; Khamboonruang, 1991; Limsuwan and with 100 muscle larvae. One month after inoculation,

Siriprasert, 1994; Khumjui et al., 2008; Kusolsuk et al., the mice were sacriﬁced and the hind limb, abdominal

2010). The species involved have been reported to be T. and diaphragm muscles were collected. Portions of the

◦

spiralis (Pozio and Khamboonruang, 1989), T. pseudospi- muscle samples were stored at −20 C for DNA extrac-

ralis (Jongwutiwes et al., 1998) and T. papuae (Khumjui tion. The remaining muscle samples were digested with

et al., 2008). In addition, the ﬁrst imported case of non- pepsin–HCl, and muscle larvae were harvested using

encapsulated T. papuae from a patient who had worked a modiﬁed Baermann technique (Justus and Morakote,

in Malaysia has been described (Chotmongkol et al., 2005; 1981). Animal experiments of this study were approved

Intapan et al., 2011). by the Animal Ethics Committee of Khon Kaen University

Molecular techniques for taxon identiﬁcation of and were based on the Ethics of Animal Experimentation

Trichinella have been developed and include the conven- of the National Research Council of Thailand (Reference

tional polymerase chain reaction (PCR), as well as the No. 0514.1.12.2/70).

multiplex PCR (Dick et al., 1992; Bandi et al., 1993, 1995;

Gasser et al., 1998; Wu et al., 1998, 1999; Zarlenga et al., 2.2. DNA extraction

1999). The development of real-time PCR has greatly

improved the molecular detection and the differential Individual and pooled muscle larvae and 50 mg

diagnosis of microorganisms within the same genus. This of infected muscle tissues of each Trichinella species

technique has increasingly replaced conventional PCR, were homogenized with disposable polypropylene pestles

which uses agarose gels and other post-PCR detection (Bellco Glass Inc., Vineland, NJ). These homogenized sam-

methods that are not as precise, are time-consuming, have ples were subsequently subjected to DNA extraction using

less sensitivity and increase the risk of amplicon contam- the NucleoSpin tissue kit (Macherey-Nagel GmbH & Co.,

ination (Zarlenga and Higgins, 2001). Effective real-time Düren, Germany). The DNA was eluted in 50 ␮l of distilled

PCR is not only accurate, sensitive, fast, and able to quan- water, and 1 ␮l of this elution was used for real-time FRET

tify speciﬁc DNA in the biological sample, but it can also PCR.

differentiate the species or strains of many medically

important pathogenic microorganisms by melting curve 2.3. Real-time ﬂuorescence resonance energy transfer

analysis (Menard et al., 2005; Hakhverdyan et al., 2006; polymerase chain reaction (real-time FRET PCR)

Abdelbaqi et al., 2007).

Recently, SYBR Green-based (Guenther et al., 2008) The real-time PCR reactions were set up in glass cap-

and Taqman probe-based real-time PCR methods (Atterby illaries and performed using a LightCycler PCR detection

et al., 2009) have been shown to be sensitive and speciﬁc for system (LightCycler 2.0, Roche Applied Science, Mannheim,

the detection of Trichinella larvae in animal muscle tissue Germany). The following genus-speciﬁc primers were

and for species differentiation. Furthermore, these tech- used: TSMito F (5 -AAT AGT GTG CCA GCT ATC G-3 ) and

niques can be used concurrently. However, in contrast to TSMito R (5 -TTA GGG GGT AAT TAG CGA GG-3 ) (Sigma-

recent reports (Guenther et al., 2008; Atterby et al., 2009), Proligo, Singapore). In addition, one adjacent oligoprobe

the use of real-time ﬂuorescence resonance energy trans- labeled at the 5 end with the LightCycler Red 640 ﬂuo-

fer (FRET) PCR with melting curve analysis has not been rophore (Tspp LC 640; 5 Red 640–GAT ACC CTT CTA TCC

feasible for the differentiation and detection of Trichinella TAG ACC TAA ACT AAT CAA GAA G–Phosphate 3 ) and

spp. The aim of the present study was to develop the real- another labeled at the 3 end with 530 ﬂuorescein (Tspp FL

time FRET PCR technique and a melting curve analysis for 530; 5 -ACA TCT GAA CTA CCA AAA GTT AAA CAA GAA

the detection and differential diagnosis of T. papuae from ACA AGG A–Fluo 530 3 ; TIB Molbiol, Berlin, Germany)

212 C. Tantrawatpan et al. / Veterinary Parasitology 185 (2012) 210–215

Fig. 1. Schematic diagram of the speciﬁc primers (TSMito F and TSMito R primers) and detection probes used for the detection of the small subunit of the

mitochondrial ribosomal RNA gene of Trichinella spp. The probe Tspp FL 530 was labeled with 530 ﬂuorescein at the 3 end and served as an anchor probe

for the sensor Tspp LC 640 probe. The sensor probe was labeled with the LightCycler Red 640 ﬂuorophore (LC Red 640) at the 5 end. The open and closed

circles represent 530 ﬂuorescein and LC Red 640, respectively. The small subunit of the mitochondrial ribosomal RNA genes of T. paupae (GenBank Accession

number EF517130), T. pseudospiralis (GenBank Accession number EF517123) and T. spiralis (GenBank Accession number AF293969) were targeted.

were used. Each glass capillary contained the PCR 2.6. Sensitivity and speciﬁcity analyses

mixture in a total volume of 20 ␮l. The mixture included

the 1 LightCycler FastStart DNA Master Hyprobe (Roche For the evaluation of analytical sensitivity, 1 ␮l of

␮

Applied Science), 3 mM MgCl2 and 0.2 M of the TSMito F genomic DNA extracted from a single larva of T. spiralis,

primer, the TSMito R primer, the Tspp FL 530 probe and the T. papuae or T. pseudospiralis and 5 ␮l of control plasmid

Tspp LC 640 probe. The PCR ampliﬁcation conditions con- T. papuae DNA were analyzed. The copy numbers ranged

◦ 5

sisted of 45 cycles of repeated denaturation (10 s at 95 C), from 1.6 to 1.6 × 10 copies. An analytical speciﬁcity test

◦ ◦

␮

annealing (30 s at 50 C) and extension (15 s at 72 C). The was performed using 10 ng of parasite genomic DNA 1 l.

◦

temperature transition rate was 20 C/s. The ampliﬁcation The panel of heterologous parasites used for testing of

◦

program was followed by a melting curve program at 95 C the real-time FRET PCR include: Ascaris lumbricoides, Cap-

◦ ◦ ◦

for 10 s, 48 C for 20 s and the transition from 48 C to 85 C illaria philippinensis, Centrocestus spp., Clonorchis sinensis,

◦

at a rate of 0.2 C/s with continuous monitoring of the ﬂu- Echinostoma malayanum, Fasciola gigantica, Giardia lamblia,

orescence. Haplorchis taichui, hookworm, intestinal lecithodendriid

ﬂukes, Isospora belli, Opisthorchis viverrini, Paragonimus

heterotremus, Schistosoma mekongi, Stellantchasmus spp.,

Strongyloides stercoralis, Taenia spp., Trichostrongylus spp.,

2.4. Primer design

and Trichuris trichiura. In addition, genomic DNA from

human leukocytes and uninfected mouse muscles were

According to the LC probe design software (Roche

included. The DNA samples used in this study were from

Applied Science), the TSMito F and TSMito R primers and

specimen banks of the Department of Parasitology, the Fac-

Tspp LC 640 and Tspp FL 530 probes were designed to

ulty of Medicine, Khon Kaen University, Thailand.

bind to the small subunit of the mitochondrial riboso-

mal RNA gene of T. papuae (GenBank Accession number

EF517130). A schematic diagram of the primers and

3. Results

hybridization probes used for these analyses is shown in

Fig. 1.

The real-time FRET PCR analysis showed positive results

for T. spiralis, T. papuae and T. pseudospiralis genomic DNA

using a 35 cycles as the cut-off between positivity and neg-

2.5. Positive control plasmids ativity. Furthermore, the species differentiation of these

three Trichinella species was based on melting tempera-

Positive control plasmids containing T. spiralis, T. pseu- ture (Tm). The mean Tm SD values for T. spiralis, T. papuae

± ±

dospiralis and T. papuae DNA were constructed by cloning and T. pseudospiralis were 51.79 0.06, 66.09 0.46 and

the 289 bp PCR product of the small subunit of the mito- 51.46 0.09, respectively (Fig. 2A). Thus, this procedure

chondrial ribosomal RNA gene into the pGEM -T Easy could detect and differentially diagnose T. papuae from T.

vector (Promega, WI, USA) according to the manufacturer’s spiralis and T. pseudospiralis. In addition, the technique was

instructions. The PCR products were obtained from con- able to detect genomic DNA extracted from larvae or in tis-

ventional PCRs using the TSMito F and TSMito R primers. sue samples from mice infected with T. spiralis (Fig. 2B), T.

The recombinant plasmids were propagated in Escherichia papuae (Fig. 2C) or T. pseudospiralis (Fig. 2D), while non-

coli. For conﬁrmation and validation of sequence informa- infected mouse tissue produced negative results.

␮

tion, the nucleotide sequences of the inserted genes were This method could use as little as 1 l of DNA extracted

␮

sequenced in both directions. from each muscle larva. Provided that 50 l of DNA was

C. Tantrawatpan et al. / Veterinary Parasitology 185 (2012) 210–215 213

Fig. 2. A representative melting curve analysis of two ﬂuorophore-labeled probes hybridized to the ampliﬁcation product of Trichinella spiralis, T. papuae

and T. pseudospiralis. The melting curves from this experiment are shown as −(d/dT) ﬂuorescence (640/530). (A) Melting peaks from 1 ␮l of eluted DNA

× 7 from one larva of T. spiralis (a), T. papuae (b) and T. pseudospiralis (c); (B) melting peaks from the T. spiralis plasmid control (5.1 10 copies) (e), genomic

DNA (68.4 ng) (f) and infected tissue (g); (C) melting peaks from T. papuae genomic DNA (16.4 ng) (i), plasmid control (1.6 × 10 copies) (j) and infected

× 7

tissue (k); (D) melting peaks from T. pseudospiralis-infected tissue (l), plasmid control (1.7 10 copies) (m) and genomic DNA (4 ng) (n). Negative controls

containing no DNA (d) and controls from uninfected mouse tissue (h) were included in this study.

prepared from each larva, then the method could detect presence of a 289-bp band upon the ampliﬁcation of T.

the equivalent of 1/50 of a single muscle larva of T. spi- spiralis, T. papuae and T. pseudospiralis DNA, whereas the

ralis, T. papuae or T. pseudospiralis (Fig. 2A). Furthermore, control genomic DNA was ampliﬁed and the non-speciﬁc

the ampliﬁcation limit was found to be approximately 16 PCR products were revealed (Fig. 4). However, no ﬂuores-

copies (Fig. 3). Real-time FRET PCR products revealed the cence signal was detected upon ampliﬁcation of DNA of

A. lumbricoides, hookworm, T. trichiura, C. philippinensis, S.

stercoralis, Trichostrongylus spp., Taenia spp., O. viverrini, H.

taichui, C. sinensis, Centrocestus spp., Stellantchasmus spp., P.

heterotremus, S. mekongi, F. gigantica, E. malayanum, intesti-

nal lecithodendriid ﬂukes, G. lamblia, I. belli or genomic

DNA from human leukocytes or uninfected mouse muscles

(Supplementary Fig. S1).

4. Discussion

In Southeast Asia and particularly in Thailand, most

human outbreaks of trichinellosis are caused by encap-

sulated T. spiralis or non-encapsulated T. papuae and

T. pseudospiralis (Pozio et al., 2009). Artiﬁcial diges-

tion is the deﬁnitive method used for the detection of

Trichinella larvae in meat. However, neither this method

nor trichinoscopy can detect enough of the small numbers

of larvae to identify Trichinella species (Forbes et al., 2003).

Recently, the real-time FRET PCR technique and the melting

Fig. 3. The ampliﬁcation plots of ﬂuorescence (y-axis) vs. cycle num-

curve analysis of speciﬁc hybridization probes and ampli-

ber (x-axis) show the analytical sensitivity of the real-time FRET PCR for

5 con products have been developed as diagnostic tools for

detecting T. papuae plasmid DNA by copy number at 1.6 × 10 copies (A),

4 3 2 species discrimination of many pathogens, including Brugia

1.6 × 10 copies (B), 1.6 × 10 copies (C), 1.6 × 10 copies (D), 16 copies (E)

and 1.6 copies (F). Distilled water was used as the negative control (G). malayi and Brugia pahangi which are the causative agents

214 C. Tantrawatpan et al. / Veterinary Parasitology 185 (2012) 210–215

Fig. 4. A 1.5% agarose gel showing the ampliﬁcation products from the analytical speciﬁcity testing by real-time FRET PCR with a 1 kb Plus DNA ladder

(Invitrogen, Carlsbad, CA) (M). (A–C) N: negative control containing no DNA; TS: T. spiralis; TP: T. papuae; TPs: T. pseudospiralis; lane 1: Fasciola gigantica;

lane 2: Paragonimus heterotremus; lane 3: Stellantchasmus spp.; lane 4: Echinostoma malayanum; lane 5: Hookworm; lane 6: Strongyloides stercoralis; lane

7: Clonorchis sinensis; lane 8: Opisthorchis viverrini; lane 9: Schistosoma mekongi; lane 10: Centrocestus spp.; lane 11: Haplorchis taichui; lane 12: Taenia spp.;

lane 13: Ascaris lumbricoides; lane 14: Trichuris trichiura; lane 15: Trichostrongylus spp.; lane 16: Giardia lamblia, lane 17: Isospora belli; lane 18: Capillaria

philippinensis; lane 19: intestinal lecithodendriid ﬂukes; lane 20: human leukocytes and lane 21: uninfected mouse tissue.

of ﬁlariasis (Thanchomnang et al., 2010), genotypes 6 and a continuous and diffuse myopathy of T. pseudospiralis-

11 of human papillomavirus (HPV) (Kocjan et al., 2010) and infected muscle cells (Wu et al., 2001; Boonmars et al.,

Campylobacter jejuni and Campylobacter coli (Abu-Halaweh 2005).

et al., 2005). In addition, the real-time FRET PCR used in this study

This study focused on the development of this assay for was able to detect as little as 1/50 the genomic DNA

the detection of T. spiralis, T. papuae and T. pseudospiralis, as extracted from one larva of each Trichinella species and

these are the indigenous species in Thailand. To differenti- approximately 16 copies of positive control plasmid. Fur-

ate between T. papuae from T. spiralis and T. pseudospiralis, thermore, this method was speciﬁc for T. spiralis, T. papuae

a melting curve analysis consisting of type-specific ampli- or T. pseudospiralis DNA; no specific fluorescence signal was

fication followed by a probe-specific post-amplification found for other parasites (Supplementary Fig. S1), although

dissociation analysis was used. The mean Tm SD val- non-speciﬁc ampliﬁed products were found. To our knowl-

ues for T. spiralis, T. papuae and T. pseudospiralis were edge, this is the ﬁrst study to use real-time FRET PCR to

51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. detect and distinguish T. papuae from T. spiralis and T. pseu-

The probes were designed to detect the sequence of the dospiralis.

small subunit of the mitochondrial ribosomal RNA gene In conclusion, the new techniques of real-time FRET PCR

of T. papuae, which varies by 7 and 9 nucleotides from and melting curve analysis are highly speciﬁc and sensitive

the sequences of T. spiralis and T. pseudospiralis, respec- for the detection and differentiation of T. papuae from T. spi-

tively. As this difference results in different Tm values, this ralis and T. pseudospiralis DNA. Because this real-time FRET

method was able to discriminate clearly between the non- PCR was able to detect muscle larvae, this method could

encapsulated larvae of T. papuae and T. pseudospiralis and also be used as a diagnostic tool for surveillance and epi-

the encapsulated larvae of T. spiralis, which was especially demiological surveys of infected wildlife in endemic areas

useful for areas in which these species are synanthropic. or for the inspection of meat in food safety programs.

However, the Tm values for T. spiralis and T. pseudospiralis

were notably similar, which caused the species discrim-

ination to be difﬁcult. However, the following biological Acknowledgments

properties and characteristics can be used to differentiate

T. spiralis and T. pseudospiralis: (1) the collagenous capsule We would like to thank Penchom Janwan and Oranuch

of T. pseudospiralis larvae is poorly developed and cannot be Sanpool for their excellent technical support. This research

identiﬁed by light microscopy (Boonmars et al., 2004); (2) was supported by grants from the National Science and

muscle cells infected with T. spiralis contain regions where Technology Development Agency (Discovery Based Devel-

the damage caused by the larvae is sealed off, whereas opment Grant), the Higher Education Research Promotion

this separation is not formed in T. pseudospiralis-infected and National Research University Project of Thailand at the

muscle cells which the entired length of the infected mus- Ofﬁce of the Higher Education Commission and the Khon

cle cell being affected (Wu et al., 2001) or (3) there is Kaen University, Thailand.

C. Tantrawatpan et al. / Veterinary Parasitology 185 (2012) 210–215 215

Appendix A. Supplementary data Jongwutiwes, S., Chantachum, N., Kraivichian, P., Siriyasatien, P., Puta-

porntip, C., Tamburrini, A., La Rosa, G., Sreesunpasirikul, C., Yingyourd,

P., Pozio, E., 1998. First outbreak of human trichinellosis caused by

Supplementary data associated with this arti-

Trichinella pseudospiralis. Clin. Infect. Dis. 26, 111–115.

cle can be found, in the online version, at Justus, D.E., Morakote, N., 1981. Mast cell degranulation associated with

doi:10.1016/j.vetpar.2011.09.043. sequestration and removal of Trichinella spiralis antigens. Int. Arch.

Allergy Appl. Immunol. 64, 371–384.

Khamboonruang, C., 1991. The present status of trichinellosis in Thailand.

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