Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
권호 표지
DOI QR코드

DOI QR Code

Development and validation of a PCR method to discriminate between Branchiostegus japonicus and Branchiostegus albus

옥돔과 옥두어 판별을 위한 PCR 검사법 개발과 검증

  • Kim, Na-Ye-Seul (Department of Food science and Technology, Suncheon National University) ;
  • Yang, Ji-Young (Department of Food Science and Technology, Pukyong National University) ;
  • Kim, Jung-Beom (Department of Food science and Technology, Suncheon National University)
  • Received : 2019.03.04
  • Accepted : 2019.04.12
  • Published : 2019.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

We developed and validated species-specific primers for Branchiostegus japonicus and Branchiostegus albus to prevent the sale of B. albus as B. japonicus. Primers for B. japonicus and B. albus were designed against the cytochrome b gene. Multiplex PCR showed a 288 bp amplicon for B. japonicus, a 159 bp amplicon for B. albus, and a 502 bp amplicon for the internal control. The PCR product bands for B. japonicus, B. albus, and the internal control were present at 1 ng each. The specificity and sensitivity of the primers developed in this study were validated by testing 38 B. japonicus strains and 13 B. albus strains. Using this monitoring method, fake fish did not appear due to the agreement between the experimental results and the species. Therefore, the developed multiplex PCR method was suitable for differentiating B. japonicus and B. albus.

본 연구에서는 형태학적으로 판별이 어려운 옥돔과 옥두어의 종 특이 primer를 개발, 검증한 후 모니터링을 통해 위변조 된 옥돔의 유통을 예방하고자 하였다. 옥돔과 옥두어 염기서열은 clustal omega 프로그램을 이용하여 정리하였고, primer3 프로그램을 이용하여 primer를 설계하였다. Multiplex PCR 결과는 옥돔과 옥두어에 대한 종 특이적 증폭이 확인되었고, PCR 반응을 확인하기 위한 공통 유전자에 대한 증폭이 확인되었다. 옥돔을 288 bp, 옥두어를 159 bp, 공통 유전자를 502 bp로 증폭되어 각각 PCR product 사이에 100 bp 이상 차이가 나타나 정확하게 판별이 가능하였다. Multiplex PCR 민감도 실험결과 옥돔 primer가 1 ng, 옥두어 primer가 1 ng, 공통 유전자 primer가 1 ng까지 밴드가 확인되었다. 모니터링 실험결과, 옥돔 38건, 옥두어 13건으로 판정되어 시료의 어종과 실험결과가 100% 일치함을 확인하였고, 위변조 사례는 나타나지 않았다. 본 실험에서 개발된 multiplex PCR 방법은 특이도와 민감도가 확보되었고 모니터링을 통해 유통, 판매되고 있는 옥돔과 옥두어의 판별에 적합함을 확인하였다.

Keywords

SPGHB5_2019_v51n3_295_f0001.png 이미지

Fig. 1. Multiplex PCR specificity of B. japonicus and B. albus.

SPGHB5_2019_v51n3_295_f0002.png 이미지

Fig. 2. Multiplex PCR sensitivity of B. japonicus and B. albus.

Table 1. Primer sequence designed in this study

SPGHB5_2019_v51n3_295_t0001.png 이미지

Table 2. Monitoring results of B. japonicus and B. albus on the market

SPGHB5_2019_v51n3_295_t0002.png 이미지

References

  1. Aida AA, Che MYB, Wong CMVL, Raha AR, Son R. Analysis of raw meats and fats of pig using polymerase chain reaction for Halal authentication. Meat Sci. 69: 47-52 (2005) https://doi.org/10.1016/j.meatsci.2004.06.020
  2. Axayacatl RO, Juan PCG. Molecular identification of dolphinfish species (genus Coryphaena) using multiplex haplotype-specific PCR of mitochondrial DNA. Ichthyol. Res. 55: 389-393 (2008) https://doi.org/10.1007/s10228-008-0040-7
  3. Choi JK, Kim HJ, Park CB, Lee CH, Song YB, Lee KJ, Yeo IK, Lee JU, Chang DS, Ha DS, Lee YD. Annual reproductive cycle and sexual characteristics of horsehead Branchiostegus japonicus. Korean J. Ichthyol. 16: 282-294 (2004)
  4. Chung IY, Seo YB, Yang JY, Kim GD. Development and validation of real-time PCR to determine Branchiostegus japonicus and B. albus species based on mitochondrial DNA. J. Life Sci. 27: 1331-1339 (2017) https://doi.org/10.5352/JLS.2017.27.11.1331
  5. Civera T. Species identification and safety of fish products. Vet. Res. Commun. 27: 481-489 (2003) https://doi.org/10.1023/B:VERC.0000014205.87859.ab
  6. Codex alimentarius commission (CAC). Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in foods. Available from: http://www.fao.org/fao-who-codexalimentarius/codex-texts/guidelines/en/. Accessed January 11, 2019.
  7. Esposti MD, De Vries S, Crimi M, Ghelli A, Patarnello T, Meyer A. Mitochondrial cytochrome b: evolution and structure of the protein. Biochim. Biophys. Acta. 1143: 243-271 (1993) https://doi.org/10.1016/0005-2728(93)90197-N
  8. Fabric T, Celia M, Catherine H. Food and forensic molecular identification: update and challenges. Trends in Biotechnol. 23: 359-366 (2005) https://doi.org/10.1016/j.tibtech.2005.05.006
  9. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech. 3: 294-299 (1994)
  10. Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B. 270: 313-321 (2003) https://doi.org/10.1098/rspb.2002.2218
  11. Irwin DM, Kocher TD, Wilson AC. Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32: 128-144 (1991) https://doi.org/10.1007/BF02515385
  12. Jenuth JP, Perterson AC, Fu K, Shoubridge EA. Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA. Nature Genet. 14: 146-151 (1996) https://doi.org/10.1038/ng1096-146
  13. Kim JH, Byun MJ, Ko YG, Kim SW, Kim SW, Do YJ, Kim MJ, Yoon SH, Choi SB. Phylogenetic analysis of Korean native goats based on the mitochondrial cytochrome b gene. J Animal Sci. Technol. 54: 241-246 (2012) https://doi.org/10.5187/JAST.2012.54.4.241
  14. Kim JC, Kang IK, Kim DS, Lee JH. The fishery and fishing ground environment for red horsehead (Branchiostegus japonicus) on the adjacent seas of Jeju Island. J. Kor. Soc. Fish. Tech. 42: 19-29 (2006) https://doi.org/10.3796/KSFT.2006.42.1.019
  15. Kim KH, Kim YS, Kim MR, Lee HY, Jung YK, Lee JH, Chang HS, Park YC, Kim SY, Choi JD, Jang YM. Development of species-specific primer to determine the authenticity of vegetable raw materials in food. Food Eng. Prog. 18: 419-426 (2014) https://doi.org/10.13050/foodengprog.2014.18.4.419
  16. Kim KH, Kim YS, Kim MR, Lee HY, Lee KH, Kim JH, Seong RS, Kang TS, Lee JH, Jang YM. Development of primer sets for the detection of Polygonum multiflorum, Cynanchum wilfordii and C. auriculatum. J. Fd Hyg. Safety 30: 289-294 (2015a) https://doi.org/10.13103/JFHS.2015.30.3.289
  17. Kim YU, Ryu JH. Taxonomic revision of the Genus Branchiostegus(Perciformes, Pisces) from the adjacent waters of Korea. Korean J. Ichthyol. 10: 40-48 (1998)
  18. Kim HS, Seo YB, Choi SS, Kim JH, Shin JY, Yang JY, Kim GD. Development and validation of multiplex polymerase chain reaction to determine squid species based on 16s rRNA gene. J. Fd Hyg. Safety 30: 43-50 (2015b) https://doi.org/10.13103/JFHS.2015.30.1.43
  19. Lee HM, Ha JS, Jo YH, Nam SH, Yoon BS. PCR detection method of Ascosphera apis, Aspergillus flavus for rapid identification of fungal disease in honeybee. Korean J. Apiculture 19: 139-148 (2004)
  20. Michael TM, Michael B, Gregory TR, Vogler AP. DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers. Phil. Trans R. Soc. B. 360: 1925-1933 (2005) https://doi.org/10.1098/rstb.2005.1724
  21. Ministry of food and drug safety (MFDS). Guidelines on standard procedures for preparing test methods such as food. Available from: http://www.mfds.go.kr/brd/m_210/view.do?seq=12920. Accessed January 11, 2019.
  22. Moretti VM, Truchini GM, Bellagamba F, Caprino F. Traceability issues in fishery and aquaculture products. Vet. Res. Commun. 27: 497-505 (2003) https://doi.org/10.1023/B:VERC.0000014207.01900.5c
  23. Noh ES, Kang HS, An CM, Park JY, Kim EM, Kang JH. Rapid and specific identification of genus Cynoglossus by multiplex PCR assays using species-specific derived from the COI region. J. Life Sci. 26: 1007-1014 (2016) https://doi.org/10.5352/JLS.2016.26.9.1007
  24. Park YC, Jung YH, Kim MR, Shin JH, Kim KH, Lee JH, Cho TY, Lee HJ, Lee SJ, Han SB. Development of detection method for Niphon spinosus, Epinephelus bruneus, and Epinephelus septemfasciatus using 16S rRNA gene. Kor. J. Food Sci. Technol. 45: 1-7 (2013) https://doi.org/10.9721/KJFST.2013.45.1.1
  25. Schellenberg A, Chmielus S, Schlicht C, Camin F, Perini M, Bontempo L, Heinrich K, Kelly SD, Rossmann A, Thomas F, Jamin E, Horacek M. Multielement stable isotope ratio (H, C, N, S) of honey from different european regions. Food Chem. 121: 770-777 (2010) https://doi.org/10.1016/j.foodchem.2009.12.082
  26. Song JY, Lim JH, Nam MH, Kim HG, Kim BS. Development of PCR primers for specific identification and detectionof Botrytis cinerea on tomato. Korean J. Mycology. 36: 138-143 (2008) https://doi.org/10.4489/KJM.2008.36.2.138