• The Korean Journal of Malacology
• /
• v.27 no.4
• /
• pp.387-393
• /
• 2011

The objective of this study was to develop a real-time PCR method for the rapid detection and quantification of the protozoan pathogen Perkinsus olseni using a TaqMan probe. For the standard, genomic DNA was extracted from $10^5$ in vitro-cultured P. olseni trophozoites, and then 10-fold serial dilutions to the level of a single cell were prepared. To test the reliability of the technique, triplicates of genomic DNA were extracted from $5{\times}10^4$ cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an $Exicycler^{TM}$ 96 real-time quantitative thermal block. For quantification, the threshold cycle ($C_T$) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the $C_T$ value and the log concentration of cells in the standard ($r^2$ = 0.996). Detection of DNA at a concentration as low as the equivalent of a single cell showed that the assay was sensitive enough to detect a single cell of P. olseni. The estimated number of P. olseni cells was similar to the original cell concentrations, indicating the reliability of P. olseni quantification by real-time PCR. Accordingly, the designed primers and probe may be used for the rapid detection and quantification of P. olseni from clam tissue, environmental water, and sediment samples.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.11
• /
• pp.1109-1115
• /
• 2011

A highly sensitive and specific TaqMan real-time PCR was used to quantify hepatopancreatic parvovirus (HPV) type III infections in wild broodstocks and hatchery-reared postlarvae (PL) of Fenneropenaeus chinensis. Totals of 159 and 162 wild brooders from three locations were captured, and 140 and 180 PL were obtained from seven and six commercial hatcheries in 2007 and 2008, respectively. Among the three wild broodstock groups from 2007, only 1 group showed HPV infection and 3.2% of 159 brooders were positive for HPV infection. In 2008, HPV infections were observed from all three wild broodstock groups with $1.93{\times}10^4$ copies/mg tissue of pleopods. Of 162 brooders, 26.6% were positive for HPV infection. No PL from the two hatcheries collected in 2007 showed HPV infection, and PL from the rest of the five hatcheries had up to $1.74{\times}10^6$ copies/ng of DNA, and PL from three hatcheries showed HPV infections with over 1,000 copies/ng of DNA. The PL from all seven hatcheries collected in 2008 showed up to $2.10{\times}10^5$ HPV copies/ng of DNA. PL from two hatcheries showed less than 100 copies/ng of DNA, but PL from the rest of the hatcheries showed HPV infections with over 1,000 copies/ng of DNA. These results show that HPV type III is widely distributed in Korea in addition to previously reported HPV type I, and they can be effectively detected by type-specific realtime PCR.

• Fisheries and Aquatic Sciences
• /
• v.12 no.3
• /
• pp.227-235
• /
• 2009

The marine toxic dinoflagellate Alexandrium catenella has been implicated in numerous paralytic shellfish poisoning (PSP) events in many countries. Due to difficulties in rapidly identifying A. catenella, field-based study of this species has been problematic. The present study developed a TaqMan format A. catenella-specific probe for real-time PCR assay (specific to Korean genotype) based on LSU rDNA sequence information for studying geographic and temporal distribution of the species in surface sediments and water columns of Jinhae Bay, Korea. The field survey from 2007 to 2008 revealed that A. catenella occurred in most seasons at low densities, mostly below 1 cell $mL^{-1}$, and was more abundant in spring (maximum cell density of 2 cells $mL^{-1}$) when shellfish exceed the quarantine toxin level for PSP toxins in Jinhae Bay.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2003.05a
• /
• pp.118-121
• /
• 2003

A method for detection and quantification of aceticlastic methanogens using a real-time PCR with a TaqMan probe was developed. Two sets of primers and probes targeting the family Methanosarcinaceae and Methanosaetaceae were designed by using the Ribosormal Database Project (RDP) II, and softwares for phylogenetic probe design and sequence analysis. Target-group specificity of each set of primers and probe was verified by testing DNAs isolated from pure cultures of 28 archaeal strains purchased from DSMZ. Cell numbers in the 28 archaeal cultures and in the samples from anaerobic processes were quantified using a real-time PCR with the sets of primers and probe. In conclusion, the real-time PCR assay was very specific for the corresponding target methanogenic family and was proved to be a powerful method for quantification of aceticlastic methanogens in anaerobic processes.

• Korean Journal of Ecology and Environment
• /
• v.54 no.3
• /
• pp.209-220
• /
• 2021

Pacific herring, Clupea pallasii, a keystone species with significant ecological and commercial importance, is declining globally throughout much of its range. While traditional fishing equipment methods remain limited, new sensitive and rapid detection methods should be developed to monitor fisheries resources. To monitor the presence and quantity of C. pallasii from environmental DNA (eDNA) extracted from seawater samples, a pair of primers and a TaqMan^® probe specific to this fish based on mitochondrial cytochrome b (COB) sequences were designed for the real-time PCR (qPCR) assay. The combination of our molecular markers showed high specificity in the qPCR assay, which affirmed the success of presenting a positive signal only in the C. pallasii specimens. The markers also showed a high sensitivity for detecting C. pallasii genomic DNA in the range of 1 pg~100 ng rxn^-1 and its DNA plasmid containing COB amplicon in the range of 1~100,000copies rxn^-1, which produced linear standard calibration curves (r²=0.99). We performed a qPCR assay for environmental water samples obtained from 29 sampling stations in the southeastern coastal regions of South Korea using molecular markers. The assay successfully detected the C. pallasii eDNA from 14 stations (48.2%), with the highest mean concentration in Jinhae Bay with a value of 76.09±18.39 pg L^-1 (246.20±58.58 copies L^-1). Our preliminary application of molecular monitoring of C. pallasii will provide essential information for efficient ecological control and management of this valuable fisheries resource.

• Korean Journal of Veterinary Service
• /
• v.45 no.1
• /
• pp.1-11
• /
• 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.

• Asian Pacific Journal of Cancer Prevention
• /
• v.17 no.11
• /
• pp.4917-4920
• /
• 2016

For advanced non-small-cell lung cancer (NSCLC) cases, a platinum-based regimen is the first-line chemotherapy treatment. The excision repair cross-complementing group 1 (ERCC1) plays an important role in DNA repair and has been related to resistance to platinum chemotherapy. This study aimed to investigate the effects of the ERCC1 (C118T) polymorphism on treatment response in 26 Thai advanced NSCLC patients receiving first line platinum-based chemotherapy during January to July 2015 at King Chulalongkorn Memorial Hospital (KCMH). DNA was extracted from peripheral blood lymphocytes and the single nucleotide polymorphism of ERCC1 was genotyped using a real-time PCR method with the TaqMan assay. The distribution of C/C, C/T and T/T genotypes was 57.7 %, 34.6 % and 7.7 %, respectively. The response rate to platinum-based chemotherapy in the wild type (C/C) of ERCC1 (C118T) was better than with the variant types (C/T and T/T) but the difference was not statistically significant (29.7% vs 9.1%, P=0.274). The results showed that a genetic polymorphism in ERCC1 might influence patient response to platinum-based chemotherapy. Further multicenter studies are now required to confirm the results of our study.

• Molecules and Cells
• /
• v.21 no.3
• /
• pp.411-417
• /
• 2006

We have developed a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.2
• /
• pp.335-341
• /
• 2007

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.

• Korean Journal of Exercise Nutrition
• /
• v.24 no.1
• /
• pp.24-28
• /
• 2020

[Purpose] Recent studies have demonstrated a probable association between ACE I/D polymorphism and obesity. Thus, this study aimed to investigate whether ACE I/D polymorphism influenced the susceptibly of developing obesity in Korean adults. [Methods] A total of 353 healthy Korean adults aged between 30 and 82 years were recruited, including 157 males and 196 females. Among the participants, 103 (29.2 %) were classified as normal (BMI < 23 kg/m²), 117 (33.1 %) as overweight (23 kg/m² ≤ BMI < 25 kg/m²), and 133 (37.7 %) as obese (BMI ≥ 25 kg/m²). ACE polymorphism (rs1799752) analysis was performed using the MGB TaqMan® SNP Genotyping assay with 3 types of primers and 2 types of probes. The distributions of the ACE genotypes and allele frequencies were analyzed among the three groups using the Hardy-Weinberg equilibrium, chi-square tests, and multiple regression analysis. [Results] The distribution of the ACE genotypes were as follows: normal [II: n=38 (36.9 %), ID: n=46 (36.8 %), DD: n=19 (18.4 %)], overweight [II: n=43 (36.8 %), ID: n=55 (47.0 %), DD: n=19 (16.2 %)], and obese [II: n=41 (30.8 %), ID: n=76 (57.0 %), DD: n=16 (12.0 %)]. Unexpectedly, the I allele, rather than the D allele, was common in the obese group. [Conclusion] ACE I/D polymorphism is not associated with BMI in Korean adults. Thus, it is unlikely to be a powerful candidate gene for obesity in Korean adults.