• Journal of Korean society of Dental Hygiene
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• v.17 no.2
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• pp.319-330
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• 2017

Objectives: The purpose of this study was to compare SYBR Green qPCR, TaqMan, and bacterial selective medium cultures for accurate quantitative analysis of oral microorganisms. Methods: The SYBR Green method is widely used to analyze the total amount of oral microorganisms in oral saliva. However, in this study, MTR-PCR method based on TaqMan method was performed using newly developed primers and probes. In addition, it was designed to confirm the detection agreement of bacteria among bacteria detection method. Results: As a result of MRT-PCR and SYBR Green qPCR analysis, more than 40 times (0.9-362.9 times) bacterium was detected by MRT-PCR. In addition, more bacteria were detected in saliva in the order of MRT-PCR, SYBR Green qPCR, and bacterium culture, and the results of MRB-PCR and SYBR Green qPCR showed the highest agreement. The agreement between the three methods for detecting P. intermedia was similar between 71.4 and 88.6%, but the agreement between MRT-PCR and SYBR Green qPCR was 80% for S. mutans. Among them, the number of total bacteria, P. intermedia and S. mutans bacteria in saliva was higher than that of SYBR Green qPCR method, and bacterium culture method by MRT-PCR method. P. intermedia and S. mutans in saliva were detected by MRT-PCR and MRT-PCR in 88.6% of cases, followed by the SYBR Green qPCR method (80.0%). Conclusions: The SYBR Green qPCR method is the same molecular biology method, but it can not analyze the germs at the same time. Bacterial culturing takes a lot of time if there is no selective culture medium. Therefore, the MRT-PCR method using newly developed primers and probes is considered to be the best method.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• The Plant Pathology Journal
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• v.39 no.6
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• pp.614-624
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• 2023

Botrytis cinerea is a major fungal plant pathogen that causes gray mold disease in strawberries, leading to a decrease in strawberry yield. While benzimidazole is widely used as a fungicide for controlling this disease, the increasing prevalence of resistant populations to this fungicide undermines its effectiveness. To investigate benzimidazole resistant B. cinerea in South Korea, 78 strains were isolated from strawberries grown in 78 different farms in 2022, and their EC₅₀ values for benzimidazole were examined. As a result, 64 strains exhibited resistance to benzimidazole, and experimental tests using detached strawberry leaves and the plants in a greenhouse confirmed the reduced efficacy of benzimidazole to control these strains. The benzimidazole resistant strains identified in this study possessed two types of mutations, E198A or E198V, in the TUB2 gene. To detect these mutations, TaqMan probes were designed, enabling rapid identification of benzimidazole resistant B. cinerea in strawberry and tomato farms. This study utilizes TaqMan real-time polymerase chain reaction analysis to swiftly identify benzimidazole resistant B. cinerea, thereby offering the possibility of effective disease management by identifying optimum locations and time of application.

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

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.369-374
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• 2014

Background: Colorectal cancer (CRC) is the fourth most common cause of cancer death in the world. Genetic variants in 8q24.21 including rs10505477 and rs6983267 have been hypothesized to be involved in susceptibility to CRC. This study aims to investigate the possible association between these loci and their haplotypes with CRC risk in Iranian population. Materials and Methods: Subjects were recruited from two hospitals in Tehran. The rs10505477 and rs6983267 polymorphisms were genotyped by TaqMan real time PCR using subject genomic DNA, extracted either from formalin-fixed, paraffin-embedded tissue of patients or from blood of the controls by standard methods. Results: A total of 715 subjects (380 CRC patients and 335 matched controls) were genotyped in this study. Allele and genotype analysis of the rs10505477 and rs6983267 polymorphisms by gender, age at diagnosis, tumor location, tumor grade, and tumor node metastasis (TNM) showed no significant association with CRC risk. There was a significant relationship between GG haplotype and susceptibility to age at diagnosis for both <60 and ${\geq}60$ (p=0.0005 and p=0.000004, respectively) and between GT and CRC in the age at diagnosis ${\geq}60$ (Table 3: p=0.031). The GG haplotype was less frequent in CRC patients with the age at diagnosis <60, but was more common in subjects with the age at diagnosis ${\geq}60$. Conclusions: Results of this study suggests that the rs6983267 and rs10505477 polymorphisms alone may not be relevant to CRC risk, but their GG haplotype plays a notable role in age at diagnosis of CRC in the Iranian population.