Abstract
The authors describe a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method targeting the 18S rRNA gene for the species detection of medically important trematode infecting fish and oysters, and suggest that this PCR-RFLP method based on a specific Tre-18 primer and the restriction enzymes, Acc1, Ava2, Msp1, and Hinf1, is useful for the detection of parasites in aquatic food.
Introduction
I
A variable domain (V4 region) of the nuclear small subunit (18S) rRNA gene was found to be useful for phylogenetic studies because of the consistent differences in this gene between trematode species (Dzikowaski et al., 2004). Accordingly, the present study addresses the need to develop PCR-RFLP targeting the 18S rRNA gene for the detection and genetic identification of several types of trematode metacercariae infecting aquatic food.
Materials and Methods
Sweetfish (Plecoglossus altivelis), mullet (Mugil cephalus), striped shiner (Pungtungia herzi), and oysters (Crassostrea gigas) are collected from the Sum-jin River and brackish water areas in the Republic of Korea. Those fish and oysters are a second intermediate host of foodborne parasites such as M. yokogawai, P. summa, H. continua, C. sinensis, and G. seoi. To collect metacercariae, fish and oysters were ground using a motor mixer, digested with artificial gastric juice (0.5% pepsin [1:10,000, Sigma Chemical Co., St. Louis, MO] and 0.8% HCl) at 37° for 2–3 h, and metacercariae were recovered using microscopy.
Genomic DNA of metacercariae was purified using a DNeasy kit (Qiagen, Rodgau-Juegesheim, Germany) and specifically amplified with primers (Tre-18, which is our naming) (F): 5′-GATAACGGGTAACGGGGAAT-3′, (R): 5′-AACCTCTGACTTTCGCTCCA-3′(2x PCR premix; Solgent Inc., Daejeon, Korea). The PCR condition is as follows: an initial denaturation at 95°C for 2 min, 35 amplification cycles (95°C for 20 s, 55°C for 30 s, and 72°C for 1 min), followed by one cycle at 72°C for 5 min. PCR products were sequenced using an ABI PRISM DNA analyzer (Applied Biosystems, CA). Gene alignments were performed using the Geneious Pro v5.0 program (Biomatters Ltd., Auckland, New Zealand). Subsequently, the species identification of trematodes was studied by RFLP analysis with Acc1, Ava2, Msp1, and Hinf1. Products digested with restriction enzymes for 2 h at 37°C were run on 3% agarose gel and band patterns were visualized on a UV transilluminator and photographed using a Gel documentation system (UVP Upland, CA).
Results and Discussion
In the present study, specific primers (Tre-18) designed by the authors can amplify a partial DNA sequence (733–739 bp) of the 18S rRNA gene in metacercariae of five trematode species. The amplified partial coding sequences of five trematode species were provided to NCBI. Their GenBank accession numbers and product size are as follows: G. seoi (JQ955636-9, 733 bp), C. sinensis (JQ955640-2, 739 bp), H. continua (JQ955643-6, 738 bp), P. summa (JQ955647-955651, 739 bp), and M. yokogawai (JQ955652-6, 739 bp). After the digestion with each restriction enzyme, the DNA band pattern is shown in Figure 1. Acc1 and Ava2 digested the 18S rRNA of G. seoi. In contrast, Msp1 digested 18S rRNA of C. sinensis (271 bp and 240 bp), P. summa (352 bp and 263 bp), M. yokogawai (397 bp and 240 bp), and H. continua (397 bp and 239 bp), but not that of G. seoi (Figure 1). Although Msp1 digested the 18S rRNA of trematodes, it was difficult to differentiate M. yokogawai and H. continua because of their similar digested band patterns. Hinf1 digested the DNA of four trematodes, except H. continua (110 bp, 217 bp, and 382 bp for all four types of trematode). These results suggest that Acc1 and Ava2 could be used to detect G. seoi, and that Msp1 could be used to detect the other trematodes. However, because P. summa and H. continua are often mixed infected in fish hosts such as A. flavimanus and M. cephalus, they can be also differentiated using Hinf1, which selectively digests 18S rRNA of P. summa.
Species identification of medically important trematodes using polymerase chain reaction (PCR)–fragment length polymorphism. The genomic DNA of five types of trematode was amplified with Tre-18 primer (left panel). The size of undigested 18S rRNA PCR product is 733–739 bp. The band shown represents the PCR products of (1) Clonorchis sinensis, (2) Gymnophalloides seoi, (3) Pygidiopsis summa, (4) Metagonimus yokogawai, and (5) Heterophyopsis continua. The five types of trematode were digested with Acc1, Ava 2, Msp1, or Hinf1 (right panel).
A recent increase in the numbers of infections caused by the consumption of undercooked aquatic food has been reported in Southeast and East Asia (Chai et al., 2009). The significant problem in the detection of parasites in aquatic food is to confirm the mixed infection of parasites in certain foods. For example, the sweetfish, Plecoglossus altivelis, the dace, Tribolodon sp., and the perch, Lateolabrax japonicus, serve as second intermediate hosts for M. yokogawai and H. continua (Chai et al., 2005, 2009). Brackish-water fish, such as the mullet, M. cephalus, the striped mullet, Liza haematocheila, the goby, Acanthogobius flavimanus, and the sweetfish, P. altivelis serve as second intermediate hosts for both P. summa and H. continua (Chai et al., 2009; Cho et al., 1985). Furthermore, human infection by C. sinensis is mediated by freshwater fish such as Zacco platypus, Cyprinus carpio, and P. herzi (Chai et al., 2005, 2009). Accordingly, we suggest that our genetic test of metacercariae targeting 18S rRNA can be used to differentiate these parasites that infect fish and oysters. Although the test described cannot discriminate all trematode species, we suggest that PCR-RFLP targeting 18S rRNA can discriminate medically important trematodes endemic in East and Southeast Asia.
Footnotes
Acknowledgments
This study was supported by the Korea National Institute of Health, Republic of Korea (grant no. NIH-4847-302-210-13, 2010-E54003-00).
Disclosure Statement
No competing financial interests exist.