• Journal of Korean Society on Water Environment
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• v.34 no.1
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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.

• Biomedical Science Letters
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• v.19 no.2
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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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• v.25 no.11
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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.

• Journal of Life Science
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• v.33 no.12
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• pp.1025-1035
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• 2023

In this study, we analyzed SNPs that appear between Korean and Chinese Scapharca subcrenata using the nucleotide sequence data of S. subcrenata analyzed by genotyping by sequencing (GBS). To distinguish the country of origin for S. subcrenata in Korean and Chinese, we developed a primer set as single nucleotide polymorphism (SNP) markers for quantitative real-time PCR (qPCR) analysis and validated by sequencing SNPs. A total of 180 samples of S. subcrenata were analyzed by genotyping by sequencing, and 15 candidate SNPs were selected. SNP marker selection for country of origin were identified through real-time qPCR. Insertion 1 and SNP 21 markers showed the most distinct separation between the sequence types as well as the country of origin through qPCR, with the observed amplification patterns matching the expected outcomes.. Additionally, in a blind test conducted by mixing samples of S. subcrenata at random, Insertion 1 showed 74% accuracy, 52% sensitivity, and 96% specificity, and SNP 21 showed 86% accuracy, 79% sensitivity, and 93% specificity. Therefore, the two SNP markers developed are expected to be useful in verifying the authenticity of the country of origin of S. subcrenata when used independently or in combination.

• Korean Journal of Breeding Science
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• v.43 no.5
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• pp.448-456
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• 2011

We used suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) method to screen differentially expressed genes from red-fleshed kiwifruit 'Hongyang'. As a result, the 288 clones were obtained by subcloning PCR product and 192 clones that showed positive clones on colony PCR analysis were selected. All the positive clones were sequenced. After comparisons with the NCBI/Genbank database using the BLAST search revealed that 30 clones showed sequence similarity to genes from other organisms; 10 clones showed significant sequence similarity to known genes. Among these clones, 3 clones (AcF21, AcF42 and AcF106) had sequence homology to 1-aminicyclopropane-carboxylic acid (ACC)-oxidase (ACO) that known to be related to fruit ripening. The expression patterns of differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qReal-time PCR) analysis. All the data from qReal-time PCR analysis coincide with the results obtained from RT-PCR analysis. Three clones were expressed at higher levels in 'Hongyang' than 'Hayward'. AcF21 was highly expressed in the other genes at 120 days after full bloom (DAFB) and 160 DAFB of 'Hongyang'.

• Biomedical Science Letters
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• v.22 no.3
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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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• v.39 no.4
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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.

• Journal of Life Science
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• v.29 no.6
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• pp.653-661
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• 2019

The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.

• Korean Journal of Veterinary Service
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• v.45 no.1
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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.

• Biomedical Science Letters
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• v.25 no.4
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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.