[논문]대기 입자상물질 시료의 곰팡이 메타게놈 분석을 위한 DNA 추출 및 PCR 조건 최적화

강수경; 조경숙

doi:10.48022/mbl.2212.12010

대기 입자상물질 시료의 곰팡이 메타게놈 분석을 위한 DNA 추출 및 PCR 조건 최적화
Optimization of DNA Extraction and PCR Conditions for Fungal Metagenome Analysis of Atmospheric Particulate Matter 원문보기

Microbiology and biotechnology letters = 한국미생물·생명공학회지, v.51 no.1, 2023년, pp.99 - 108

강수경 (이화여자대학교 환경공학과) , 조경숙 (이화여자대학교 환경공학과)

초록
AI-Helper

대기 입자상물질(particulate matter, PM) 시료의 곰팡이 메타게놈 분석을 위해 DNA 추출 및 유전자 증폭 시 여러 문제가 발생한다. 본 연구에서는 PM 시료로부터 DNA를 추출하는 방법과 polymerase chain reaction (PCR)을 위한 프라이머 및 온도 조건의 최적화를 위하여 다양한 조건으로 실험하였다. 여러 조건에서 DNA 추출 여부를 비교 평가한 결과, bufffer와 proteinase K를 이용하여 20분 동안 화학적 세포 용해 처리와 bead beating 처리를 한 후 상용 DNA 추출 kit를 사용하면 DNA를 효율적으로 추출할 수 있었다. PCR 조건을 최적화하기 위해 ITS2 유전자 영역을 증폭할 수 있는 10개 조합의 프라이머를 이용하여 PCR을 수행한 결과, ITS3tagmix3/ITS4 조합의 프라이머로 annealing 온도 58℃로 하였을 때 증폭된 PCR 산물의 농도가 상대적으로 높았다. 이 조건에서도 PCR 산물의 농도가 낮은 경우에는 1차 PCR 산물을 주형 DNA로 사용하여 nested PCR을 수행하면 만족스러운 농도로 ITS2 유전자를 증폭할 수 있었다. 본 연구에서 도출한 조건으로 서울 대기 PM2.5를 포집한 필터 시료 15종을 대상으로 DNA 추출과 PCR을 수행한 결과 성공적으로 ITS2 유전자 증폭이 가능하였다. 본 연구에서 최적화한 방법은 대기 PM 시료의 곰팡이 메타게놈을 분석하고 해석하는 연구에 활용 가능하다.

Abstract ▼ AI-Helper

Several challenges arise in DNA extraction and gene amplification for airborne fungal metagenome analysis from a particulate matter (PM) samples. In this study, various conditions were tested to optimize the DNA extraction method from PM samples and polymerase chain reaction (PCR) conditions with primer set and annealing temperature. As a result of comparative evaluation of DNA extraction under various conditions, chemical cell lysis using buffer and proteinase K for 20 minutes and bead beating treatment were followed by using a commercial DNA extraction kit to efficiently extract DNA from the PM filter samples. To optimize the PCR conditions, PCR was performed using 10 primer sets for amplifying the ITS2 gene region. The concentration of the PCR amplicon was relatively high when the annealing temperature was 58℃ with the ITS3tagmix3/ITS4 primer set. Even under these conditions, when the concentration of the PCR product was low, nested PCR was performed using the primary PCR amplicon as the template DNA to amplify the ITS2 gene at a satisfactory concentration. Using the methods optimized in this study, DNA extraction and PCR were performed on 15 filter samples that collected PM2.5 in Seoul, and the ITS2 gene was successfully amplified in all samples. The optimized methods can be used for research on analyzing and interpreting the fungal metagenome of atmospheric PM samples.

주제어

표/그림 (7)

표 Table 1. Information of PM2.5 filter samples for repeatability evaluation.
그림 Fig. 1. Pretreatment methods for DNA extraction. (A) Slicing method. (B) Complex method. (C) Bead beating method.
그림 Fig. 2. Comparison of DNA concentrations of negative control (sterilized filter), positive control (filter inoculated with Bacillus subtilis), and PM samples without pretreatments. Sterilized, sterilized filter; Bacillus A (OD 2.324), filter with undiluted Bacillus subtilis culture broth; Bacillus B (OD 0.639), filter with diluted B. subtilis culture broth; PM2.5, filter collected particulate matters under 2.5 μm diameter; TSP, filter collected total suspended particulates.
표 Table 2. Nucleotide sequence of primers for amplifying ITS2 region.
그림 Fig. 3. Comparison of DNA concentrations by different pretreatment methods using commercial DNA extraction kits. (A) Com-mercial DNA extraction kit for soil (Soil kit). (B) Commercial DNA extraction kit for water (Water kit). Soil kit, Soil kit without pretreatment; Slicing (1/4), Slicing method + 1/4 of filter; Slicing (1), Slicing + whole filter; Complex (1/4), Complex method + 1/4 of filter; Complex (1), Complex method + whole filter; Longer lysis, Increasing chemical lysis time. Water kit, Water kit without pretreatment; Longer lysis,Increasing chemical lysis time; Longer lysis and bead beating, Increasing chemical lysis time + bead beating method.
표 Table 3. Relative abundances of electrophoresis gel bands in combination primers with annealing temperatures.
그림 Fig. 4. DNA concentrations and PCR products of PM2.5 samples using the optimized methodology. (A) DNA concen-trations. (B) Relative abundance of gel band for PCR products. (C) Electrophoresis gel pictures of PCR products.

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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