타액내 구강질환 원인 균의 세균배양법, SYBR green qPCR법, MRT-PCR법 간의 정량분석 = Quantitative analysis of oral disease-causing bacteria in saliva among bacterial culture, SYBRgreen qPCR and MRT-PCR method

다국어 초록 (Multilingual Abstract)

Objectives: The purpose of this study was to compare SYBR Green qPCR, TaqMan, and bacterial selective medium cultures for accurate quantitative analysis of oral microorganisms. Methods: The SYBR Green method is widely used to analyze the total amount of oral microorganisms in oral saliva. However, in this study, MTR-PCR method based on TaqMan method was performed using newly developed primers and probes. In addition, it was designed to confirm the detection agreement of bacteria among bacteria detection method. Results: As a result of MRT-PCR and SYBR Green qPCR analysis, more than 40 times (0.9-362.9 times) bacterium was detected by MRT-PCR. In addition, more bacteria were detected in saliva in the order of MRT-PCR, SYBR Green qPCR, and bacterium culture, and the results of MRB-PCR and SYBR Green qPCR showed the highest agreement. The agreement between the three methods for detecting P. intermedia was similar between 71.4 and 88.6%, but the agreement between MRT-PCR and SYBR Green qPCR was 80% for S. mutans. Among them, the number of total bacteria, P. intermedia and S. mutans bacteria in saliva was higher than that of SYBR Green qPCR method, and bacterium culture method by MRT-PCR method. P. intermedia and S. mutans in saliva were detected by MRT-PCR and MRT-PCR in 88.6% of cases, followed by the SYBR Green qPCR method (80.0%). Conclusions: The SYBR Green qPCR method is the same molecular biology method, but it can not analyze the germs at the same time. Bacterial culturing takes a lot of time if there is no selective culture medium. Therefore, the MRT-PCR method using newly developed primers and probes is considered to be the best method.

참고문헌 (Reference)

1 김지훈, "중합효소연쇄반응법을 이용한 급성 치수 및 치근단 질환의 병원성 세균의 동정" 대한치과보존학회 28 (28): 178-183, 2003

2 Darveau RP, "The microbial challenge in periodontitis" 14 : 12-32, 1997

3 Kyung-Hui Moon, "The Influence on the Recognition for Periodontal Care to Oral Micro-Organism Changes in Dental Implant Patients" 대한예방치과학회 12 (12): 221-228, 2016

4 Slots J, "Subgingival microflora and periodontal disease" 6 (6): 351-382, 1979

5 김민정, "Strain-specific PCR Primers for the Detection of Prevotella intermedia ATCC 49046" 대한구강생물학회 36 (36): 79-82, 2011

6 Lee JW, "Simultaneous detecting composition of multiple oral disease bacteria with multiplex real-time PCR and method thereof. Patent ․ Registration No. 1017060700000"

7 Akira Yano, "Real-time PCR for quantification of streptococcus mutans" Oxford University Press (OUP) 217 (217): 23-30, 2002

8 Martin FE, "Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis" 40 (40): 1698-1704, 2002

9 Reardon-Robinson ME, "Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development" 111 (111): 3835-3840, 2014

10 Dymock D, "Molecular analysis of micro¯ora associated with dentoalveolar abscesses" 34 : 537-542, 1996

11 Haffajee AD, "Microbial etiological agents of destructive periodontal diseases" 5 : 78-111, 1994

12 V.I. Haraszthy, "Identification of Periodontal Pathogens in Atheromatous Plaques" Wiley-Blackwell 71 (71): 1554-1560, 2000

13 H. P. Horz, "Evaluation of Universal Probes and Primer Sets for Assessing Total Bacterial Load in Clinical Samples: General Implications and Practical Use in Endodontic Antimicrobial Therapy" American Society for Microbiology 43 (43): 5332-5337, 2005

14 Gouet P, "ESPript: Analysis of multiple sequence alignments in PostScript" 15 (15): 305-308, 1999

15 Soon-Nang Park, "Development of quantitative real-time PCR primers for detecting 42 oral bacterial species" Springer Nature 195 (195): 473-482, 2013

16 박순낭, "Development of Quantitative Real-Time PCR Primers for the Detection of Aggregatibacter actinomycetemcomitans" 대한구강생물학회 36 (36): 1-6, 2011

17 Nadkarni MA, "Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set" 148 (148): 257-266, 2002

18 Mättö J, "Detection of porphyromonas gingivalisfrom saliva by PCR by using a simple sample-processing method" 36 (36): 157-160, 1998

19 Boutaga K, "Comparison of real-time PCR and culture for detection of porphyromonas gingivalis in subgingivalplaque samples" 41 (41): 4950-4954, 2003

20 P.-M. Jervoe-Storm, "Comparison of culture and real-time PCR for detection and quantification of five putative periodontopathogenic bacteria in subgingival plaque samples" Wiley-Blackwell 32 (32): 778-783, 2005

21 Raouf Wahab Ali, "Comparative Detection Frequency of 6 Putative Periodontal Pathogens in Sudanese and Norwegian Adult Periodontitis Patients" Wiley-Blackwell 65 (65): 1046-1052, 1994

22 C. Verner, "Carpegen® real-time polymerase chain reaction vs. anaerobic culture for periodontal pathogen identification" Wiley-Blackwell 21 (21): 341-346, 2006

23 Kroes I, "Bacterial diversity within the human subgingival crevice" 96 (96): 14547-14552, 1999