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PCR을 이용한 치아우식증 및 치주염 연관 병원체의 빠른 검출

Rapid Detection of Pathogens Associated with Dental Caries and Periodontitis by PCR Using a Modified DNA Extraction Method

  • 김재환 (전북대학교 치의학전문대학원 소아치과학교실 및 구강생체과학연구소) ;
  • 김미아 (전북대학교 치의학전문대학원 소아치과학교실 및 구강생체과학연구소) ;
  • 이대우 (전북대학교 치의학전문대학원 소아치과학교실 및 구강생체과학연구소) ;
  • 백병주 (전북대학교 치의학전문대학원 소아치과학교실 및 구강생체과학연구소) ;
  • 양연미 (전북대학교 치의학전문대학원 소아치과학교실 및 구강생체과학연구소) ;
  • 김재곤 (전북대학교 치의학전문대학원 소아치과학교실 및 구강생체과학연구소)
  • Kim, Jaehwan (Department of Pediatric Dentistry and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University) ;
  • Kim, Miah (Department of Pediatric Dentistry and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University) ;
  • Lee, Daewoo (Department of Pediatric Dentistry and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University) ;
  • Baik, Byeongju (Department of Pediatric Dentistry and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University) ;
  • Yang, Yeonmi (Department of Pediatric Dentistry and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University) ;
  • Kim, Jaegon (Department of Pediatric Dentistry and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University)
  • 투고 : 2014.06.16
  • 심사 : 2014.09.17
  • 발행 : 2014.11.30

초록

구강 병원체의 검출 방법은 여러 가지가 있지만 그 중 PCR을 이용한 검출이 확실하고 빠른 방법으로 알려져 있다. PCR을 위한 많은 DNA 추출법이 사용되고 있으나 상업적인 DNA 추출 kit들은 일반적으로 가격이 비싸고 절차가 여러 단계로 되어있으며, 그 외의 방법은 페놀과 클로로포름과 같은 유해한 화학물질을 써야하는 등의 단점이 있다. 이 연구에서 NaOH 용액을 이용한 개선된 DNA 추출 방법은 치아우식증, 치주염과 관련된 병원체를 빠르고 간단하며 비용-효율적으로 검출하였다. 세균으로부터 DNA를 추출하기 위한 boiling은 기존의 10분이 아닌 1분으로 충분하였고 $4^{\circ}C$에서 최소 13개월 이상 DNA의 보관이 가능하였으며 sonication 유무에 따른 차이는 없었다. 따라서, 이 방법은 상업적인 kit나 유해한 화학물질을 쓰지 않고서도 타액 표본으로부터 직접적으로 빠른 시간 내에 DNA를 추출하여 병원체의 유무 결과를 확인하는데 매우 적합할 것으로 생각한다.

DNA extraction is a prerequisite for the identification of pathogens in clinical samples. Commercial DNA extraction kits generally involve time-consuming and laborious multi-step procedures. In the present study, our modified DNA isolation method for saliva samples allows for the quick detection of pathogens associated with dental caries or periodontitis by PCR within 1 h. To release DNA from the bacteria, 1 min of boiling was adequate, and the resulting isolated DNA can be used many times and is suitable for long term storage of at least 13 months at $4^{\circ}C$, and even longer at $-20^{\circ}C$. In conclusion, our modified DNA extraction method is simple, rapid, and cost-effective, and suitable for preparing DNA from clinical samples for PCR for the rapid detection of oral pathogens from saliva.

키워드

참고문헌

  1. Watanabe K : Prepubertal periodontitis: A review of diagnostic criteria, pathogenesis, and different diagnosis. J Periodontal Res, 25:31-48, 1990. https://doi.org/10.1111/j.1600-0765.1990.tb01205.x
  2. Watanabe K, Frommel TO : Detection of Porphyromonas gingivalis in oral plaque samples by use of the polymerase chain reaction. J Dent Res, 72:1040-1044, 1993. https://doi.org/10.1177/00220345930720060801
  3. Lin CY, Wong MY, Kuo MY, et al. : Rapid and specific detection of the leukotoxin sequences of Actinobacillus actinomycetemcomitans from periodontal pockets by the polymerase chain reaction. J Formosan Med Assoc, 93:289-293, 1994.
  4. Slots J, Ashimoto A, Chen C, et al. : Detection of putative periodontal pathogens in subgingival specimens by 16S ribosomal DNA amplification with the polymerase chain reaction. Clin Infec Dis, 20:304-307, 1995. https://doi.org/10.1093/clinids/20.Supplement_2.S304
  5. Wahlfors J, Meurman JH, Vaisanen P, et al. : Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a rapid PCR method. J Dent Res, 74:1796-1801, 1995. https://doi.org/10.1177/00220345950740111301
  6. Okada M, Hayashi F, Nagasaka N : Detection of Actinobacillus actinomycetemcomitans and porphyromonas gingivalis in dental plaque samples from children 2 to 12 years of age. J Clin Periodontol, 27:763-768, 2000. https://doi.org/10.1034/j.1600-051x.2000.027010763.x
  7. Okada M, Soda Y, Hayashi F, et al. : PCR detection of Streptococcus mutans and S. sobrinus in dental plaque samples from Japanese pre-school children. J Med Mcrobiol, 51: 443-447, 2002. https://doi.org/10.1099/0022-1317-51-5-443
  8. Shiroza T, Shinozaki N, Abiko Y, et al. : Rapid isolation of chromosomal DNA from oral streptococci and polymerase chain reaction-oriented restriction fragment-length polymorphism analysis for genetic heterogeneity. Oral Microbiol Immunol, 13:11-16, 1998. https://doi.org/10.1111/j.1399-302X.1998.tb00744.x
  9. Smith GL, Socransky SS, Smith CM : Rapid method for the purification of DNA from subgingival microorganisms. Oral microbial Immunol, 4:47-51, 1989. https://doi.org/10.1111/j.1399-302X.1989.tb00406.x
  10. Parrish KD, Greenberg EP : A rapid method for extraction and purification of DNA from dental plaque. Appl Environ Microbiol, 61:4120-4123, 1995.
  11. An investigation of a rapid DNA extraction method for routine MAS in the S.A. Barley Improvement Program. Available from URL: http://www.regional.org.au/au/abts/2001/t4/warner.htm (assessed on April 21, 2014)
  12. Saarela M, Hannula J, Alaluusua S, et al. : Typing of mutans streptococci by arbitrarily primed polymerase chain reaction. Arch Oral Biol, 41:821-826, 1996. https://doi.org/10.1016/S0003-9969(96)00049-0
  13. Yano A, Kaneko N, Hanada N, et al. : Real-time PCR for quantification of Streptococcus mutans, FEMS Microbiol Lett, 217:23-30, 2002. https://doi.org/10.1111/j.1574-6968.2002.tb11451.x
  14. Eisenach KD, Cave MD, Crawford JT, et al. : Detection of Mycobacterium tuberculosis in clinical sputum samples using a polymerase chain reaction. Am Rev Respir Dis, 144:1160-1163, 1991. https://doi.org/10.1164/ajrccm/144.5.1160

피인용 문헌

  1. Clinical Assessment and Survey of Periodontal Condition among Adolescents vol.43, pp.3, 2016, https://doi.org/10.5933/JKAPD.2016.43.3.227