Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
권호 표지
DOI QR코드

DOI QR Code

Real-time PCR assay for the Detection of Toxoplasma gondii in Retail Meats: Proof-of-concept Study

유통식육에서의 톡소포자충 검출을 위한 유전자검사법 개발

  • Yun, Han Seong (Food Microbiology Division, Ministry of Food and Drug Safety) ;
  • Suh, Soo Hwan (Food Microbiology Division, Ministry of Food and Drug Safety) ;
  • Kwak, Hyo-Sun (Food Microbiology Division, Ministry of Food and Drug Safety) ;
  • Joo, In-Sun (Food Microbiology Division, Ministry of Food and Drug Safety)
  • 윤한성 (식품의약품안전처 미생물과) ;
  • 서수환 (식품의약품안전처 미생물과) ;
  • 곽효선 (식품의약품안전처 미생물과) ;
  • 주인선 (식품의약품안전처 미생물과)
  • Received : 2017.02.20
  • Accepted : 2017.04.24
  • Published : 2017.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Although many PCR-based assays have been developed, the majority of rapid detection of Toxoplasma gondii in animal and their meat product has been dependent on immunogenic assays. Thus, there is still a need for more reliable PCR based detection method for T. gondii in retail meats. Recently, a 529-bp repeat element that exists in 200-300 copies per genome of T. gondii genome had been spotlighted for its usefulness as potential detection targers. In this study, the 529-bp repeat element was selected for real-time PCR to detect three types of T. gondii (type I, II and III). A primer pair targeting 82-bp of the 529-bp element detected all three types of T. gondii and showed high level of specificity against 14 different food-borne pathogens as well as 3 protozoan parasites such as Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica. Application of the new real-time PCR assay in meat samples showed improved detection sensitivity compared to the B1-gene targeted method suggesting potential new target for Toxoplasma gondii screening in retail meats.

인수공통 감염증의 하나인 톡소포자충의 검출을 위해서는 대부분은 ELISA 법이 사용 되고 있으나, 충체가 사멸된 후에도 양성반응이 나타나는 등 사용에 제한이 있다. 반면 유전자 검출법은 현재 감염상태를 확인 할 수 있기 때문에 식중독 원인조사 등에 적합하다고 판단되어 이를 활용하여 본 연구를 진행하였다. 톡소포자충의 유전정보를 통해 529 repeat region의 염기서열을 얻고, 프라이머 및 TaqMan 프로브를 설계하여 real-time PCR을 이용한 검출법을 개발하였다. 검출한계(lower limit of detection) 및 적정곡선을 확인한 결과 10 genomic DNA copy가 검출한계로 확인되었고, 정량을 위한 곡선은 $10^1{\sim}10^6$ DNA copies까지 0.999의 $R^2$ 값을 나타내었다. 개발된 검출법의 증폭효율을 비교하기 위해 B1 gene 타겟 프라이머 세트와 타입별 검출한계를 비교한 결과, type 1, 2, 3 톡소포자충에서 같거나 더 나은 검출한계를 보였다. 또한 식품에서 주로 분리되는 식중독 세균 14종 및 원충 3종에 대해 특이도를 비교한 결과, 모두 음성으로 나타났다. 개발된 검출법을 식육검체에 적용하였을 때 type 1, 2, 3에서 모두 원활한 검출결과를 보여 증폭방해물질이 존재하지 않은 것으로 확인되었다. 본 연구를 통해 개발된 유전자검출법은 국내 유통 중인 식육에서 인수 공통감염 원충의 하나인 톡소포자충의 감염 여부를 확인하는 사전적 모니터링의 방법으로 활용될 예정이다.

Keywords

References

  1. Al-Karmi T., Behbehani K.: Epidemiology of toxoplasmosis in Kuwait. II. Toxoplasma gondii in the desert rodent, Meriones crassus. Trans R Soc Trop Med Hyg, 74, 745-746 (1980). https://doi.org/10.1016/0035-9203(80)90191-1
  2. Bartova E., Sedlak K., Literak I.: Prevalence of Toxoplasma gondii and Neospora caninum antibodies in wild boars in the Czech Republic. Vet Parasitol, 142, 150-153 (2006). https://doi.org/10.1016/j.vetpar.2006.06.022
  3. Buchbinder S., Blatz R., Rodloff A.C.: Comparison of realtime PCR detection methods for B1 and P30 genes of Toxoplasma gondii. Microbiol Infect Dis, 45, 269-271 (2003). https://doi.org/10.1016/S0732-8893(02)00549-7
  4. Burg J.L., Grover C.M., Pouletty P., Boothroyd J.C.: Direct and Sensitive Detection of a Pathogenic Protozoan Toxoplasma gondii, by Polymerase Chain Reaction. J. Clin. Microbiol, 1787-1792 (1989).
  5. Cazabonne P., Bessieres M.H., Seguela J.P.: Kinetics study and characterization of target excreted/secreted antigens of immunoglobulin G, M, A and E antibodies from mice infected with different strains of Toxoplasma gondii. Parasittol Res, 80, 58-63 (1994). https://doi.org/10.1007/BF00932625
  6. Cheong K.S., An S.C., Kim J.O., Kim N.S., Chang K.H.: A studies on toxoplasmosis antibody from slaughtered pigs and cattle. Korean J Vet Serv, 17, 32-36 (1994).
  7. Dubey J.P.: Advances in the life cycle of Toxoplasma gondii. Int J Parasitol, 28, 1019-1024 (1998). https://doi.org/10.1016/S0020-7519(98)00023-X
  8. Dubey J.P. and Beattie C.P.: Toxoplasmosis of animal and Man. CRC press Inc, Florida, 1-213 (1998).
  9. Dubey J.P., Hill D.E., Jones J.L., Hightower A.W., Kirkland E., Roberts J.M., Marcet P.L., Lehmann T., Vianna M.C.B., Miska K., Sreekumar C., Kwok O.C.H., Shen S.K., Gamble H.R.: Prevalence of viable Toxoplasma gondii in beef, chicken and pork from retail meat stores in the United States: risk assessment to consumers. J. Parasitol, 91, 1082-1093 (2005). https://doi.org/10.1645/GE-683.1
  10. Grigg M.E.and Boothroyd J.C.: Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene. J Clin Microbiol, 39, 398-400 (2001). https://doi.org/10.1128/JCM.39.1.398-400.2001
  11. Homan W.L., Vercammen M., De Braekeleer J., Verschueren H.: Identification of a 200 to 300 fold repetitive 529bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. J Parasitol, 30, 69-75 (2000).
  12. Howe D.K. and Sibley L.D.: Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human diseases. J Infect Dis, 172, 1561-1566 (1995). https://doi.org/10.1093/infdis/172.6.1561
  13. Israelski D.M. and Remington J.S.: Toxoplasmosis in patients with cancer. Clin Infect Dis, 47, 413-415 (1993).
  14. Jones C.D., Okbravi N., Adamson P., Tasker S., Ligbtman S.: Comparison of PCR Detection Methods for B1, P30, and 18S rDNA Genes of T. gondii in Aqueous Humor. Investigative Ophthalmology & Visual Science, 416, 34-644 (2000).
  15. Jones J.L. and Dubey J.P.: Foodborne Toxoplasmosis. Clin Infect Dis, 55, 845-851 (2012). https://doi.org/10.1093/cid/cis508
  16. Kim E.G., Park H.J., Son G.B., Jung M.H.: Prevalence of Toxoplama gondii infection from domestic pig in Gyeongnam province. Korean J Vet Serv, 33, 345-351 (2010).
  17. Kim N.H., Kim H.R., Park H.S., Kim Y.S., Lee JH.: Seroprevalence of Coxiella burnetii and Toxoplasma gondii in cattle in Seoul, Korea. Korean J Vet Serv, 38, 233-239 (2015). https://doi.org/10.7853/kjvs.2015.38.4.233
  18. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J., Higgins DG.: Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948 (2007). https://doi.org/10.1093/bioinformatics/btm404
  19. Lee Y.H., Kim K.Y., Kang M.S., Shin D.W.: Detection of Toxoplasma antigens and antibodies in mice infected with different strains of Toxoplasma gondii. Korean J. Parasitol, 33, 201-210 (1995). https://doi.org/10.3347/kjp.1995.33.3.201
  20. Lin M.H., Chen T.C., Kuo T.T., Tseng C.C., Tseng C.P.: Real-Time PCR for Quantitative Detection of Toxoplasma gondii. J. Clin. Microbiol, 38, 4121-4125 (2000).
  21. Lukesova D. and Literak I.: Shedding of Toxoplasma gondii oocysts by Felidae in zoos in the Czech Republic. Vet Parasitol, 74, 1-7 (1998). https://doi.org/10.1016/S0304-4017(97)00155-6
  22. Mastroianni A., Coronado O., Scarani P., Manfredi R., Chiodo F.: Anergic disseminated toxoplasmosis in a patient with AIDS. Minerva Med, 88, 101-104 (1997).
  23. Montoya J.G. and Liesenfeld O.: Toxoplasmosis. Lancet 363,1965-1976 (2004). https://doi.org/10.1016/S0140-6736(04)16412-X
  24. Neva F.A. and Brown H.W.: Basic & Clinical Parasitology, 6th ed. Norwalk: Appliton & Lange. 46-56 (1994).
  25. Racka K., Bartova E., Budikova M., Vodrazka P.: Survey of Toxoplasma gondii antibodies in meat juice of wild boar (Sus scrofa) in several districts of the Czech Republic. Ann Agric Environ Med, 14, 71-73 (2007).
  26. Romero C.R., Buitron G.R., Amancio C.O., Tay Z.J., Sanchez V.J.T.: Toxoplasmosis and threatened abortion. Ginecol Obstet Mex, 66, 495-498 (1998).
  27. Seo M.G., Jang Y.S., Lee E.M., Park N.C., Kwak D.M.: Prevalence of antibodies to Toxoplasma gondii in cattle and pigs reared in eastern areas of Gyeongbuk province. Korean J. Vet Serv, 32, 131-137 (2009).
  28. Sherdering G.R.. Toxoplasmosis and other sysemic protozoal infection. In: Saunders Manual of small animal practice, 3rd ed, Ohio, 219-225 (1996).
  29. Shin S.S.: Pathgenicity and prevention of swine toxoplasmosis. Kor J Vet Publ Hlth, 21, 173-181 (2007).
  30. Song K.J., Shin J.C., Shin H.J., Nam H.W.: Seroprevalence of toxoplasmosis in Korean pregnant women. Korean J parasitol, 43, 69-71 (2005). https://doi.org/10.3347/kjp.2005.43.2.69
  31. Silva J.C., Ogassawara S., Adania C.H., Ferreia F., Gennari S.M., Dubey J.P., Ferreira-Neto J.S.: Seroprevalence of Toxoplasma gondii in captive neotropical felids from Brazil. Vet Parasitol, 102, 217-224 (2001). https://doi.org/10.1016/S0304-4017(01)00523-4
  32. Silva J.C., Ogassawara S., Marvulo M.F., Ferreira-Neto J.S., Dubey J.P.: Toxoplasma gondii antibodies in exotic wild felids from Brazilian zoos. J Zoo Wildl Med, 32, 349-351 (2001). https://doi.org/10.1638/1042-7260(2001)032[0349:TGAIEW]2.0.CO;2
  33. Tenter A.M., Heckeroth A.R., Weiss L.M.: Toxoplasma gondii: from animals to humans. Int J Parasitol, 31, 217-20 (2001). https://doi.org/10.1016/S0020-7519(01)00125-4
  34. Villena I., Aubert D., Gomis P., Ferte H., Inglard J.C., Denis B.H., Dondon J.M., Pisano E., Ortis N., Pinon J.M.: Evaluation of a strategy for Toxoplasma gondii oocyst detection in water. Appl Environ Microbiol, 70, 4035-4039 (2004). https://doi.org/10.1128/AEM.70.7.4035-4039.2004