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 Method원문보기
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.
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 ...
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.
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.
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문제 정의
12) and it has simple steps to prepare genomic DNA and does not use toxic reagents. The purpose of this study was to investigate the efficiency of the modified DNA extraction method for the preparation of genomic DNA from saliva samples and identification of oral pathogens.
제안 방법
Phenol is a hazardous organic compound that causes chemical burns and chloroform is also considered hazardous when it is inhaled since it can depress central nervous system and cause death when it is severe. The DNA extraction procedure used in this study was modified from Saarela et al.12) and it has simple steps to prepare genomic DNA and does not use toxic reagents. The purpose of this study was to investigate the efficiency of the modified DNA extraction method for the preparation of genomic DNA from saliva samples and identification of oral pathogens.
대상 데이터
Saliva samples were collected by spitting saliva into a 50 ml sterile tube from 5 patients (aged between 9 and 12 years old) who visited Dental Hospital of Chonbuk National University.
성능/효과
The pathogens were well detected by PCR using the specific primers(Fig. 3) and the PCR results performed with the DNA main-tained for 13 months showed no difference from the first one. It took less than 5 min to prepare genomic DNA from each sample using the modified DNA extraction method and the DNA was well amplified by PCR.
A study suggested that the DNA extracted using NaOH was quick but easily degradable and could not be stored more than 1 month at 4℃11). However, the DNA we isolated using the modified extraction method in this study was stable and in a good condition for PCR for at least 13 months at 4℃ and it could be maintained for a longer term at -20℃. For the intention of disrupting Gram-positive bacteria more easi-ly, sonication procedure was added to the method.
We stored the DNA samples separate-ly at 4℃ and -20℃ to investigate whether temperature affected maintenance and degradation of them since storage temperature could influence DNA and make it not sufficient for PCR. However, the result of the PCR with DNA stored at 4℃ in this study showed that tem-perature does not have much impact on it for PCR for at least 13 months(Fig. 2) and the DNA could be used many times (data not shown).
Oral pathogens can be de-tected within 1 hour by PCR using the modified DNA method. In conclusion, the data of this study shows that the modified DNA extraction method used in this study is simple, rapid, and cost-efficient.
참고문헌 (14)
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