Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit

Kang, Minji,Yang, Jeong Seong,Kim, Yoojeong,Kim, Kyungjong,Choi, Hongjo,Lee, Seung Heon Elsevier 2018 Journal of microbiological methods Vol.152 No.-

<P><B>Abstract</B></P> <P>Tuberculosis (TB), caused by infections of the <I>Mycobacterium tuberculosis</I> (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by <I>N</I>-acetyl-<SMALL>L</SMALL>-cysteine-2% NaOH and then extracted using three methods: “in-house” 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Magnetic beads extract DNA of greater purity than that extracted by resin. </LI> <LI> Magnetic bead DNA extraction had higher efficiency in detection of TB genes. </LI> <LI> The automated extraction system is technically simple and completely closed. </LI> <LI> Magnetic bead extraction is optimal for anti-TB drug susceptibility gene testing. </LI> </UL> </P>

인삼의 진세노사이드 분석을 위한 추출 및 전처리법

김금숙,현동윤,김영옥,이성우,김영창,이승은,손영득,이민정,박충범,박호기,차선우,송경식 한국약용작물학회 2008 한국약용작물학회지 Vol.16 No.6

An advanced extraction method by ultrasonic extraction with applied solid phase extraction (SPE) has been developed for the determination of simultaneous eight major ginsenosides, namely ginsenosides Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, and Rd in the root of Panax ginseng. Four extraction methods including n-BuOH reflux extraction (Method A), 70% EtOH reflux extraction (Method B), 50% MeOH reflux extraction with SPE (Method C), and 50% MeOH ultrasonication with SPE clean-up process (Method D) were investigated for the determination of eight major ginsenosides. Total contents of ginsenosides were highest by extraction of Method C as 2.408±0.011%. However, Method D was evaluated as relatively simpler and more efficient method due to short extraction time, small solvent consumption and less expensive, compared to conservative reflux method. Ginsenosides were also satisfactorily separated with good resolution and the accuracy range was between 1.05 and 4.06% as relative standard deviation (RSD) by Method D. SPE condition and HPLC condition were further optimized for determination of eight major ginsenosides by the ultrasonic extraction method. Conclusively, ultrasonic extraction of 2 g sample of ginseng using ultrasonic bath and 1 loading for SPE was evaluated as proper condition for extraction of ginseng.

전탕 방법 및 전탕 시간에 따른 십전대보탕 전탕액 비교 연구

김정훈 ( Jung Hoon Kim ),서창섭 ( Chang Seob Seo ),전우영 ( Woo Young Jeon ),신현규 ( Hyeun Kyoo Shin ) 대한한방부인과학회 2012 大韓韓方婦人科學會誌 Vol.25 No.2

Objectives: This study was performed to compare the differences between decoctions extracted by different extraction method and extraction time. Methods: Decoctions were prepared with pressurized or non-pressurized extraction for 60, 120 and 180min. The yield of extract, total soluble solid content, hydrogen ion concentration(pH) and the content of reference compound in Sipjeondaebo-tang (Siquandabu-tang) were investigated. Results: While yields and the total soluble solid of decoction were higher in pressurized method proportional to extraction time, pH values were lower in pressurized method and showed decreasing values with increasing extraction time. Albilflorin, ferulic acid, nodakenin, coumarin, cinnamaldehyde and glycyrrhizin were contained in decoctions by pressurized extraction method more than non-pressurized method whereas the content of coumarin was higher in decoctions by non-pressurized extraction method. In addition, coumarin was extracted increasingly with extraction time in decoctions by pressurized method, however, nine compounds except cinnamaldehyde showed the tendency of increasing proportional to extraction time. Conclusions: The results show that extraction methods including pressurized or non-pressurized extraction, and extraction time could affect the physicochemical characteristic or composition of Sipjeondaebo-tang(Siquandabu-tang) decoction.

전탕 방법 및 전탕 시간에 따른 육미지황탕 전탕액 비교 연구

김정훈,서창섭,전우영,신현규,Kim, Jung-Hoon,Seo, Chang-Seob,Jeon, Woo-Young,Shin, Hyeun-Kyoo 대한한의학방제학회 2011 大韓韓醫學方劑學會誌 Vol.19 No.1

Objectives : This study was performed to compare the differences between decoctions extracted by different extraction method and extraction time. Methods : Decoctions were prepared with pressed or non-pressed extraction conditions for 60, 120 and 180min. The yields of extracts, sugar contents, hydrogen ion concentrations(pH), the contents of reference compounds in Yukmijihwang-tang(Liuweidihuang-tang) were investigated. Results : The yields and the dissolved solid content containing sugar content of decoctions were more in pressed extraction method than unpressed extraction method, and they tended to be increased as extraction time increased. The pH values of decoctions methods did not show significant differences between pressed and unpressed extraction methods or extraction times. Most of reference compounds showed higher contents in pressed extraction method than unpressed extraction method and their contents were augmented according to increase of extraction time. The content of paeonol was decreased when extracted in more than 120min with pressed extraction method and tended to be decreased as extraction time increased in unpressed extraction method. Conclusions : The pressed extraction with long extraction time could be useful for decoction of Yukmijihwang-tang(Liuweidihuang-tang). However, another ingredients possible to decrease in such condition need to be considered to determine suitable extraction condition.

Extraction Methods of Organic Components from Rubber Composites and Analysis of the Extract Using Gas Chromatography/Mass Spectrometry

( Eunji Chae ),( Sung-seen Choi ) 한국고무학회 2019 엘라스토머 및 콤포지트 Vol.54 No.3

Rubber articles contain various organic additives such as antidegradants, curing agents, and processing aids. It is important to extract and analyze these organic additives. In this paper, various extraction methods of organic additives present in rubber composites were introduced (solvent extraction, Soxhlet extraction, headspace extraction, and solid-phase microextraction), and the extracts were characterized using gas chromatography/mass spectrometry (GC/MS). Solvent and Soxhlet extractions are easy-to-perform and commonly used methods. Efficiency of solvent extraction varies according to the type of solvent used and the extraction conditions. Soxhlet extraction requires a large volume of solvent. Headspace sampling is suitable for extracting volatile organic compounds, while solid-phase extraction is suitable for extracting specific chemicals. GC/MS is generally used for characterizing the extract of a rubber composite because most components of the extract are volatile and have low molecular weights. Identification methods of chemical structures of the components separated by GC column were also introduced.

금은화의 폴리페놀, 플라보노이드, Chlorogenic acid의 최적 추출조건

송선영,김명기,하헌용 조선대학교 공학기술연구원 2018 공학기술논문지 Vol.11 No.1

This study was carried out to compare of extraction efficiency for Lonicera Flos by extraction conditions. To compare the extraction efficiency of active ingredients by the extraction methods and ratio of water to ethanol (0, 20, 40, 60, 80 and 100% water) for Lonicera japonica Flos was extracted by ultrasonic extraction, shaking extraction water-bath extraction, and soaking extraction. Contents of phenolic compounds were the highest with 127.9 mg GAE/g DW in 60% ethanol by hot-water extraction method at 80℃ and contents of flavonoids were highest with 29.8 mg GAE/g DW in 80% ethanol by hot-water extraction method at 80℃. Contents of chlologenic acid were the highest with 31.8 mg/g in 80% ethanol by hot-water extraction method at 80℃. Extraction of the bioactive ingredient of Lonicera japonica Flos was effective in 60-80% ethanol by hot-water extraction.

국제표준(안) 개선을 위한 토양 중 화약물질 추출 효율성 평가

이군택,정인호,김보현,김동욱,Lee, Goon-Taek,Jung, In-Ho,Kim, Bo-Hyun,Kim, Dong-Wook 한국지하수토양환경학회 2012 지하수토양환경 Vol.17 No.1

The ISO document of ISO/TC/190/SC3/WG11/N11 is a working draft of international standard (WD) dealing with analytical method for the determination of explosives and related compounds using high performance liquid chromatography. The scope of this WD covers the storage of samples, preparing test portion, extraction and instrumentation. The main purpose of this study was to improve the extraction conditions which were already adopted in the WD. For this purpose, mechanical shaking method could be corresponded up to 18 hours of ultrasonic bath extraction in the WD was tested. Methanol was also tested with the intention of being added as an extracting solvent other than acetonitrile in the WD. According to the results, 16 hours of mechanical shaking method showed statistically the same effectiveness as that of 18 hours of ultrasonic bath extraction. In case of extracting solvent, methanol also showed statistically the same extraction capability as acetonitrile for DNB, TNT, 2-A-DNT and 2,4-DNT. However, the recovery rate of TNB with methanol extraction was 40% higher than that of acetonitrile extraction. Through adding mechanical shaking method into committee draft (cf. the next stage draft of the WD during the process for making international standard), ISO standard of analyzing explosives and related compounds in soil would become more useful in dealing with huge number of field samples in the laboratory. In other aspect, adopting methanol as an alternative extracting solvent would be very effective in the terms of exchangeability with GC-ECD/MS method which is being developed by German experts.

인삼의 추출조건 및 진세노사이드의 HPLC 분석법 평가

이경희,이대영,이승은,남기열,김형돈,이재원,최재훈,안영섭,김승유,김금숙,황광보 한국약용작물학회 2016 한국약용작물학회지 Vol.24 No.1

Background : A new extraction method-heated ultrasonic extraction was qualitatively and quantitatively analyzed for the extraction of major ginsenosides from ginseng extract; this new high-performance liquid chromatography (HPLC) method was compared with the official extraction method of Korean industrial standards and standard for health functional food. Methods and Results : Ginsenoside compounds were analyzed for 35 minutes by the new HPLC analysis method using a Halo® RP-Amide column. The new HPLC analysis method was validated by the measurement of intra-day and inter-day precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) of each ginsenoside. The correlation coefficients (r2) for the calibration curves of the ginsenoside compounds were over 0.9997 in terms of linearity. The heated ultrasonic extraction method using ultrasonication for 30 minutes at 50℃ yielded higher amount of ginsenosides than the extraction method of the Korean industrial standards owing to the enhancement of extraction efficiency. Conclusions : Compared to the other extraction methods, the heated ultrasonic extraction method yielded a higher amount of ginsenoside Rb1 than Rg1 index compounds for the quality evaluation of ginseng roots.

추출방법을 달리한 달맞이꽃 추출물의 항산화 및 항균 활성

김진학,이신호 한국식품저장유통학회 2016 한국식품저장유통학회지 Vol.23 No.2

A effect of extraction methods, including stirrer extraction method (SE), ultrasonification extraction method (USE), reflux extraction method (RE), autoclave extraction (AE) and low temperature high pressure extraction (LE) method on the antioxidative and antimicrobial activities of ethanol extracts of Oenothera biennis was investigated. The extraction yield (46.33%), total polyphenol (463.05 mg GAE/g) and flavonoid (71.71 mg RHE/g) content of Oenothera biennis extract obtained by RE were higher than those from other extraction methods. The antimicrobial activity of Oenothera biennis extract was only observed against Bacillus cereus among other tested organisms (Bacillus cereus, Staphylococcus aureus, Escherichia coli and Salmonella Typhimurium). Oenothera biennis obtained by RE showed the best DPPH radical scavenging ability (74.40%), ABTS radical scavenging ability (65.29%), reducing power (1.370 (OD700)) and ferrous ion chelating ability (90.14%) compared with other tested extraction methods tested. The RE method was the most efficient method for extracting crude antioxidant and antimicrobial substances from Oenothera biennis. These results suggested that Oenothera biennis obtained by RE could be used as a bioactive and functional material in the food industry.

노니 부정근으로부터 유효성분의 최적 추출조건

김명기(Myong-Ki Kim) 한국식품과학회 2016 한국식품과학회지 Vol.48 No.2

노니 부정근 유효성분의 효율적인 추출조건을 규명하고자 용매 종류, 물과 메탄올의 비율(물, 20, 40, 60, 80, 100%), 추출시간 및 추출방법을 달리하여 실험을 진행하였다. 안트라퀴논, 페놀성 화합물 및 플라보노이드의 용매별 추출한 결과 1시간 동안 초음파 추출 시 60-80% 메탄올에서 효과적이었으며, 환류냉각 추출기로 2시간 추출 시 80% 메탄올에서 효과적이었다. 노니 부정근의 유효성분에 대한 추출방법 및 시간에 따른 추출효율을 비교하기 위하여 교반추출, 환류냉각추출, 초음파추출, 균질기추출, 초고압추출, 침지추출을 실시하였다. 페놀성 화합물은 초음파추출에서 가장 높은 함량을 보였으며 안트라퀴논과 플라보노이드 함량은 환류냉각추출에서 가장 높게 나타났다. To determine the optimum condition for the extraction of active components, noni adventitious roots were extracted under various conditions employing various solvents including methanolic water at different ratio (20, 40, 60, 80, and 100% water), extraction times and extraction methods. Anthraquinones, phenolic compounds, and flavonoids extraction using different solvents showed that 1 h of ultrasonic extraction was effective in 60-80% methanol, and 2 h of reflux extraction was effective in 80% methanol. To compare the extraction efficiency of active components according to different extraction methods and extraction times for noni adventitious roots, the active components were extracted by ultrasonic extraction, shaking extraction, reflux extraction, homogenizer extraction, high-pressure extraction, and soaking extraction. The highest phenolic contents were found in the extracted from ultrasonic extraction and anthraquinones and flavonoids contents were highest in the reflux extraction.