• 한국약용작물학회지
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• 제18권1호
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• pp.40-45
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• 2010

The application of PCR analysis on the herbal medicine Moutan Cortex (Paeonia suffruticosa Andrews) was evaluated by the comparison of the genetic relationship based on the DNA sequence with Paeoniae Radix (Paeonia lactiflora Pallas) following development of specific primers. Moutan Cortex and Paeoniae Radix were distinguished through the PCR analysis based on the internal transcribed spacer (ITS-PCR) from nuclear ribosomal DNA region. The 294 bp PCR products both of Moutan Cortex and Paeoniae Radix was amplified by MIF1 and MIR1. And a Moutan Cortex specific 225 bp PCR amplification product was amplified by MIF2 and MIR1 primers. The 225 bp sequence could be successfully amplified from Mortan Cortex of dried herbal preparations. PCR analysis based on ITS (ITS-PCR) may be an efficient tool for the discrimination of Moutan Cortex.

• 분석과학
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• 제12권3호
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• pp.260-264
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• 1999

법적시료(Forensic Evidences)로 사용되는 혈흔, 정액반, 타액반 및 모발을 DNA의 분리과정 없이 FoLT (Formamide Low Temperature) PCR법으로 DNA의 특정부위를 분석하고자 하였다. FoLT PCR법으로 3종류의 STR(Short Tandem Repeat) 좌위와 성별을 확인하는 Amelogenin gene을 PCR법으로 동시에 분석하기 위하여 formamide농도와 annealing온도를 검토한 바 최적의 formamide농도는 8%(v/v)이었고 annealing온도는 $48^{\circ}C$이었다. 그리고 1% Triton X-100을 이용하여 시료를 세척한 경우에 증폭 효율이 증가하였다. 따라서 FoLT-PCR법을 이용한 경우 DNA를 분리하기 위한 전처리없이 소량의 시료를 기존의 PCR법보다 빠른 시간내에 한번의 PCR 및 전기영동으로 HumTH01, HumTPOX, HumCSF1PO 및 Amelogenin을 동시에 분석할 수 있었다.

• Applied Biological Chemistry
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• 제50권4호
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• pp.245-252
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• 2007

FTA(fault tree analysis)는 system 오류 관리를 위한 정성적/정량적 기법으로 적용되고 있다. FTA를 적용한 PCR의 오류 관리 system의 구축을 위한 시범적 단계로서 PCR 실행의 여러 단계 중 가장 간단한 단계인 '반응액의 제조 및 PCR 기기 사용 단계'를 모델로 하여 분석하였다. PCR 실행시 발생할 수 있는 오류를 연역적 논리 방식에 의해 fault tree의 형태로 규명하였다. Fault tree는 오류 관리의 최상위 요소인 top event를 중심으로 중간 계층을 이루는 intermediate events와 최하위의 요소인 basic events로 세분하여 구성하였다. Top event는 '반응액의 제조 및 PCR 기기 사용 단계에서의 오류'; 중간계층 events는 '기기 유래 오류', '실험행위 유래 오류'; basic events는 '정전상황', 'PCR 기기 선정', '기기 사용 관리', '기기 내구성', '조작의 오류', '시료 구분의 오류'로 분석되었다. 이로부터 top event의 원인 분석 및 중요 관리점을 도출하기 위하여 정성적/정량적 분석을 실시하였다. 정성적 기법으로 minimal cut sets, structural importance, common cause vulnerability를 분석하였고, 정량적 기법으로 simulation, cut set importance, item importance, sensitivity를 분석하였다. 정성적 분석과 정량적 분석의 결과에서 '시료 구분의 오류'와 '기기 조작의 오류'가 제 1중요관리점; '기기 관리의 오류'와 '내구성에 의한 오류'는 제 2중요관리점으로 일치되게 나타났다. 그러나 '정전상황'과 '기기 선정의 오류'는 정성적 분석에서만 중요관리점으로 분석되었다. 특히 sensitivity 분석에서 '기기 관리의 오류'는 사용 시간이 경과함에 따라 가장 중요한 관리점으로 부각되었다. 결론적으로 FTA는 PCR 모델 case에 대한 오류의 원인 분석 및 그 방지를 위한 중요관리점을 제시함에 따라, 궁극적으로 미래에 PCR의 오류 관리 system을 완성할 수 있는 효과적인 방법으로 사료된다.

• 공업화학
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• 제33권3호
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• pp.235-241
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• 2022

유엔이 발표한 세계 물개발 보고서는 2030년까지 식수가 현재보다 40% 감소할 것으로 전망하고 있다. 이는 물의 양이 감소하는 것이 아니라, 환경오염으로 인해 상수원이 오염되는 것을 말한다. 미생물이 수질에 깊은 연관이 있기 때문에 미생물의 분석은 수질관리에 매우 중요하다. 현재 미생물 분석에 사용되는 방법은 배양 후 현미경을 통한 모양과 형태를 분석하는 것이 가장 일반적이나, 유전자분석 기술이 발달함에 따라 현미경을 통한 미생물 분석 방식에 qPCR(quantitative polymerase chain reaction) 적용이 가능해졌고 활용방법 등이 연구되었다. 그 중에는 역전사 단계를 추가하여 RNA 분석에 용이성을 부여한 RT-qPCR법과 미생물 배양분석에 접목시켜 검사시간을 단축시키는 ICC-qPCR, 자연에서 채취한 샘플의 위양성율을 감소시키는 데 용이한 viability qPCR, 다중분석에 용이한 multiplex qPCR, 소량의 샘플만으로 분석이 가능한 microfluidic qPCR법 등이 있다. 본 논문에서는 이처럼 qPCR 방법이 미생물 분석에 적용되는 사례와 방식의 원리, 그리고 발전 방향에 대해 소개하고자 한다.

• Journal of Microbiology
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• 제44권2호
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• pp.155-161
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• 2006

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63 % (137 of 217) in the standard PCR method to about 34 % (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: ${\alpha},\;{\beta},\;{\gamma}-$ Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

• Parasites, Hosts and Diseases
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• 제55권6호
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• pp.601-606
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• 2017

Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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• pp.358-367
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• 2021

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

• Genomics & Informatics
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• 제21권2호
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• 대한의생명과학회지
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• 제9권3호
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• pp.139-144
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• 2003

Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

• 환경생물
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• 제22권2호
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.