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Differential Detection of Trichinella Papuae, T. Spiralis and T Veterinary Parasitology 185 (2012) 210–215 Contents lists available at SciVerse ScienceDirect 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 a Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen 40002, Thailand b Division of Cell Biology, Department of Preclinical Sciences, Thammasat University, Rangsit Campus, Pathum Thani 12121, Thailand c Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand d Faculty of Medicine, Mahasarakham University, Mahasarakham 44150, Thailand e Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand f Department of Parasitology, Gifu University Graduate School of Medicine, Yanagido1-1, Gifu 501-1194, Japan g 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 infected humans and animals. © 2011 Elsevier B.V. All rights reserved. ∗ 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). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.09.043 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.
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