• 한국물환경학회지
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• 제34권1호
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• pp.46-56
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• 2018

A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

• 대한의생명과학회지
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• 제19권2호
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• pp.153-157
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• 2013

Although culture is the gold standard method to identify mycobacteria, its use in tuberculous lymphadenitis (TBL) is limited due to formalin fixation of the submitted specimens. We evaluated the performance of quantitative real-time PCR (q-PCR) for Mycobacterium Tuberculosis (MTB) in granulomatous lymphadenitis using formalin-fixed paraffin-embedded (FFPE) tissues. From 2000 to 2010, a total number of 117 cases of lymph node samples with granulomatous inflammation which were surgically removed and fixed in formalin were studied. Hematoxylin & Eosin (H&E) and Ziehl-Neelsen-stained (ZN) slides were reviewed. qPCR using Real TB-Taq$^{(R)}$ was performed for all cases to identify Mycobacterium tuberculosis. Thirteen non-tuberculous lymphadenopathy cases were used as negative control. Cervical lymph nodes were more frequently affected (60%, 70/117) than other sites. ZN stain for acid fast bacilli was positive in 19 (16.24%) cases. qPCR for tuberculosis was positive in 92 (78.63%) cases. Caseous necrosis was found in 103 (88.03%) cases. While the ZN stain and qPCR were both negative in all control cases, the qPCR showed a significantly higher positive rate (78.63% vs. 16.24%) compared to ZN stain in histologically diagnosed TBL. Quantitative real-time PCR proves to be more sensitive than ZN stain for diagnosis of tuberculous lymphadenitis.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1928-1935
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• 2015

Microbial growth kinetics is often used to optimize environmental processes owing to its relation to the breakdown of substrate (contaminants). However, the quantification of bacterial populations in the environment is difficult owing to the challenges of monitoring a specific bacterial population within a diverse microbial community. Conventional methods are unable to detect and quantify the growth of individual strains separately in the mixed culture reactor. This work describes a novel quantitative PCR (qPCR)-based genomic approach to quantify each species in mixed culture and interpret its growth kinetics in the mixed system. Batch experiments were performed for both single and dual cultures of Pseudomonas putida and Escherichia coli K12 to obtain Monod kinetic parameters (μ_max and K_s). The growth curves and kinetics obtained by conventional methods (i.e., dry weight measurement and absorbance reading) were compared with that obtained by qPCR assay. We anticipate that the adoption of this qPCR-based genomic assay can contribute significantly to traditional microbial kinetics, modeling practice, and the operation of bioreactors, where handling of complex mixed cultures is required.

• 생명과학회지
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• 제33권12호
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• pp.1025-1035
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• 2023

본 연구에서는 한국산과 중국산 새꼬막(Scapharca subcrenata) 사이의 원산지 판별을 위하여 quantitative real-time PCR (qPCR) 분석을 기반으로 하는 Single-nucleotide polymorphism (SNP) 마커의 primer set를 개발 및 검증하였다. 총 180개의 새꼬막 sample을 genotyping by sequencing으로 분석하여 원산지 판별에 유용할 것이라 판단되는 7개의 후보 MS 마커와 15개의 후보 SNP 마커를 선정하였다. 후보 SNP 마커는 PCR과 sanger sequencing, SYBR green-based qPCR을 통해 원산지별 분리 여부를 확인하였다. 이 중 Insertion 1, SNP 21 마커가 qPCR 증폭 양상에서 집단이 확연히 분리되었으며 예상과 실제 증폭 형태가 일치하였다. 추가적으로 새꼬막을 무작위로 섞어서 진행한 blind test에서 Insertion 1은 새꼬막 100개에 대하여 74%의 정확도, 52%의 민감도, 96%의 특이도를 보였고, SNP 21은 새꼬막 137개에 대하여 86%의 정확도, 79%의 민감도, 93%의 특이도를 보였다. 따라서 개발된 두 개의 SNP 마커는 독립적 또는 복합적으로 사용하면 새꼬막 원산지 판별의 진위 여부를 검증하는 데 유용할 것으로 기대된다.

• 한국육종학회지
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• 제43권5호
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• pp.448-456
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• 2011

적색 과육 '홍양' 품종에서 차등발현하는 유전자를 찾기 위하여 mirror orientation selection (MOS)과 결합된 suppression subtractive hybridization (SSH) 실험을 수행하였다. 그 결과, 288개의 cDNA clone을 확보하였으며, colony PCR을 통해 192개의 positive clone을 선발하였고, 이들을 sequencing하였다. NCBI/Genbank 데이터베이스의 BLAST 검색를 이용하여 염기서열을 분석한 결과, 30개의 clone에서 기존에 알려진 유전자기능과의 유사성을 확인할 수 있었으며, 10개의 clone이 특이유전자였다. 그 중 3개의 clone(AcF21, AcF42, AcF106)은 과실 후숙과 관련된 ACC-oxidase와 상동성이 있었다. SSH의 결과를 통해 얻어진 이 유전자들의 차등발현양상을 확인하기 위하여 reverse transcription PCR(RT-PCR)과 quantitative real-time PCR(qReal-time PCR) 분석을 실시하였다. qReal-time PCR분석과 RT-PCR분석에서 모두 동일한 결과를 확인할 수 있었으며, 3개 clone 모두 '홍양'에서의 유전자발현수준이 '헤이워드'보다 더 높았다. AcF21은 다른 유전자들보다 가장 높은 발현수준을 나타내었는데, 만개 후 120일과 160일 모두 '홍양'에서의 발현수준이 높았다.

• 대한의생명과학회지
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• 제22권3호
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• pp.83-97
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• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.

• The Plant Pathology Journal
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• 제39권4호
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• 생명과학회지
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• 제29권6호
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• pp.653-661
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• 2019

본 연구는 siRNA 기반 치료제등의 핵산치료제 개발에 있어서 필수적인 약물의 생체내 흡수, 분포, 대사, 배설에 대한 동태의 확인을 위해 stem-loop RT-qPCR 법을 이용하여 보다 더 정확한 시험법을 확립하고자 하였다. siRNA에 특이적인 primer와 probe를 선별하여 siRNA 정량검출 시험법을 최적화하였다. siRNA 표준시료를 이용하여 최적화된 시험법을 적용하였을 때 siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과, 기울기 평균 -3.3, 결정계수 $R^2$>0.99으로 확인되어 siRNA 표준시료와 Cp 값 간의 회귀성이 매우 높아 정량 분석이 가능한 시험법임을 확인하였고, 같은 표준시료를 이용한 stem-loop RT-qPCR의 검출한계(LOD)는 10 fM, 최소정량한계(LLOQ)는 100 fM이었다. 확립된 시험법의 신뢰성을 확인하기 위해 시험자를 다르게 하고, 시험법을 3회 반복하여 각각 진행한 결과, siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과 기울기와 결정계수 $R^2$의 재현성(slope ${\pm}-3.2$, 결정계수 $R^2$>0.99)을 확인하였고, 표준 곡선으로부터 환산된 siRNA 표준시료의 회수율(recovery ${\pm}20%$)과 완건성이 우수함을 확인하였다. 확립된 stem-loop RT-qPCR을 생체내 존재하는 약물 검증에 적용할 수 있는지 확인하기 위하여 시험동물에 siRNA를 주입 후 시간별 혈액을 채취하여 확립된 시험법으로 시험을 진행하였고 약물 동태학적 분석을 통해 siRNA치료제의 혈액내의 안정성을 확인하였다. 따라서 본연구에서 개발된 stem-loop RT-qPCR 분석법은 정확성, 정밀성 및 민감도가 높은 분석법으로 핵산치료제 개발 연구의 다양한 생체시료 분석 연구에 적용할 수 있을 것으로 기대한다.

• 한국동물위생학회지
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• 제45권1호
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• pp.1-11
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• 2022

A novel porcine circovirus 4 (PCV4) was recently identified in Chinese and Korean pig herds. Although several conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR) assays were used for PCV4 detection, more sensitive and reliable qPCR assay is needed that can simultaneously detect PCV4 and internal positive control (IPC) to avoid false-negative results. In the present study, a duplex qPCR (dqPCR) assay was developed using primers/probe sets targeting the PCV4 Cap gene and pig (glyceraldehyde-3-phosphate dehydrogenase) GAPDH gene as an IPC. The developed dqPCR assay was specifically detected PCV4 but not other PCVs and porcine pathogens, indicating that the newly designed primers/probe set is specific to the PCV4 Cap gene. Furthermore, GAPDH was stably amplified by the dqPCR in all tested viral and clinical samples containing pig cellular materials, indicating the high reliability of the dqPCR assay. The limit of detection of the assay 5 copies of the target PCV4 genes, but the sensitivity of the assay was higher than that of the previously described assays. The assay demonstrated high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1.0%. Clinical evaluation using 102 diseased pig samples from 18 pig farms showed that PCV4 circulated in the Korean pig population. The detection rate of PCV4 obtained using the newly developed dqPCR was 26.5% (27/102), which was higher than that obtained using the previously described cPCR and TaqMan probe-based qPCR and similar to that obtained using the previously described SYBR Green-based qPCR. The dqPCR assay with IPC is highly specific, sensitive, and reliable for detecting PCV4 from clinical samples, and it will be useful for etiological diagnosis, epidemiological study, and control of the PCV4 infections.

• 대한의생명과학회지
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• 제25권4호
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• pp.407-416
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• 2019

Recent years have seen an increase in the incidence of candidiasis caused by non-albicans Candida (NAC) species. In fact, C. glabrata is now second only to C. albicans as the most common cause of invasive candidiasis. Therefore, the rapid genotyping specifically for C. glabrata is required for early diagnosis and treatment of candidiasis. A number of genotyping assays have been developed to differentiate C. glabrata sequence types (STs), but they have several limitations. In the previous study, multi-locus sequence typing (MLST) has performed with a total of 101 C. glabrata clinical isolates to analyze the prevalent C. glabrata STs in Korea. A total of 11 different C. glabrata STs were identified and, among them, ST-138 was the most commonly classified. Thus, a novel probe-based quantitative PCR (qPCR) assay was developed and evaluated for rapid and accurate identification of the predominant C. glabrata ST-138 in Korea. Two primer pairs and hybridization probe sets were designed for the amplification of internal transcribed spacer 1 (ITS1) region and TRP1 gene. Analytical sensitivity of the probe-based qPCR assay was 100 ng to 10 pg and 100 ng to 100 pg (per 1 μL), which target ITS1 region and TRP1 gene, respectively. This assay did not react with any other Candida species and bacteria except C. glabrata. Of the 101 clinical isolates, 99 cases (98%) were concordant with MLST results. This novel probe-based qPCR assay proved to be rapid, sensitive, highly specific, reproducible, and cost-effective than other genotyping assay for C. glabrata ST-138 identification.