• Research in Plant Disease
• /
• v.27 no.3
• /
• pp.120-127
• /
• 2021

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

• Toxicological Research
• /
• v.32 no.1
• /
• pp.81-87
• /
• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• Korean Journal of Veterinary Service
• /
• v.44 no.3
• /
• pp.163-168
• /
• 2021

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in isolating infected patients and preventing further viral transmission. In this study, we evaluated the clinical diagnostic performances of a commercial real-time reverse transcription loop-mediated isothermal amplification (RRT-LAMP) assay (Isopollo^® COVID-2 assay, M-monitor, Daegu, Korea) using eighty COVID-19 suspected clinical samples and compared these with the results of a commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) assay (Allplex^TM 2019-nCoV rRT-QPCR Assay, SeeGene, Seoul, Korea). The results of the RRT-LAMP assay targeting the N or RdRp gene of SARS-CoV-2 showed perfect agreement with the RT-qPCR assay results in terms of detection. Furthermore, the RRT-LAMP assay was completed in just within a 20-min reaction time, which is significantly faster than about the 2 h currently required for the RT-qPCR assay, thus enabling prompt decision making regarding the isolation of infected patients. The RRT-LAMP assay will be a valuable tool for rapid, sensitive, and specific detection of SARS-CoV-2 in human or unexpected animal clinical cases.

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

• Journal of the korean academy of Pediatric Dentistry
• /
• v.45 no.1
• /
• pp.32-40
• /
• 2018

The aim of this study was to determine the efficacy of a 3 tone plaque disclosing gel in assessing the risk of caries related to the population of Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus spp. quantified using a quantitative real-time polymerase chain reaction (qRT-PCR). 15 healthy children of ages 9 - 12 years were randomly examined. The 3 tone plaque disclosing gel was applied on teeth surfaces, which changed the color to pink or red, blue or purple and light blue. Plaque was divided into 3 groups based on staining. Genomic DNA from each sample was subjected to a qRT-PCR assay for quantitative detection of target bacteria. The Kruskal-Wallis test was conducted for correlation between the color of plaque and the number of bacterial species. The levels of S. mutans, S. sobrinus, and Lactobacillus spp. were significantly different in the plaque samples of the 3 groups (p < 0.05). The proportion of S. sobrinus to S. mutans showed correlation to the color of plaque. The different color-dyed plaque was related to the number of acidogenic bacteria. The 3 tone plaque disclosing gel could be used as one of the indicators to assess the clinical risk of caries associated with the population of S. mutans, S. sobrinus, and Lactobacillus spp.

• Korean Journal of Veterinary Service
• /
• v.45 no.4
• /
• pp.305-316
• /
• 2022

Bordetella (B.) bronchiseptica, Mycoplasma (M.) felis, and Chlamydia (C.) felis are considered as main bacterial pathogens of feline upper respiratory tract disease (URTD). In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) assay was developed for the rapid and differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with the detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1%. Based on the diagnostic results of the assay using 94 clinical samples obtained from cats with URTD signs, prevalence of B. bronchiseptica, M. felis, or C. felis was 10.6%, 36.2%, or 6.4%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported monoplex qPCR assays. The dual infection rates for B. bronchiseptica and M. felis or M. felis and C. felis was 2.1% or 3.2%, respectively. These results indicated that M. felis has been widely spread, and its co-infection with B. bronchiseptica or M. felis has been frequently occurred in Korean cat population. The developed tqPCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens and the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of feline URTD in Korea.

• Journal of the Korean Society of Marine Environment & Safety
• /
• v.26 no.6
• /
• pp.674-680
• /
• 2020

Quantitative real-time polymerase chain reaction (qPCR) was applied for the early detection of red tides in the coastal areas of South Gyeongsang in 2019. Cochlodinium polykrikoides (Dinophyceae) was detected at very low cell densities (0.0015~0.0058 cells mL^-1) in early June, but its cell density increased by up to 0.163 cells mL^-1 in mid-August. Higher cell densities were detected mainly in Namhaedo using both qPCR and microscopy (maximum 24 cells mL^-1) in late-August. Accordingly, a red tide alert was issued on September 2 (maximum 200 cells mL^-1) on this island. C. polykrikoides cell density in Namhaedo peaked on September 11 (12,000 cells mL^-1). Our results indicate that C. polykrikoides was detected at very low cell density in Namhaedo prior to bloom, which occurred in the same area. Therefore, qPCR is a useful tool to detect even at very low cell densities of C. polykrikoides for early warning of blooms.

• Korean Journal of Microbiology
• /
• v.49 no.3
• /
• pp.270-274
• /
• 2013

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.5
• /
• pp.630-636
• /
• 2013

Recently, there has been a growing body of evidence showing that cellular microRNAs (miRNAs) are involved in virus-host interactions. Numerous studies have focused on analyses of the expression profiles of cellular miRNAs, but the expression patterns of Dicer, which is responsible for the generation of miRNAs, have only rarely been explored in Gallus gallus. We developed a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the relative quantification of the mRNAs of Dicer and ${\beta}$-actin in G. gallus. To apply this method, the expression of Dicer in avian cells after infection with avian leukosis virus subgroup J (ALV-J) was detected using our established duplex real-time RT-PCR. The duplex real-time RT-PCR assay is sufficiently sensitive, specific, accurate, reproducible, and cost-effective for the detection of Dicer in G. gallus. Furthermore, this study, for the first time, demonstrated that ALV-J can induce differential expression of Dicer mRNA in the ALV-J-infected cells.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.24
• /
• pp.10585-10589
• /
• 2015

Breast cancer susceptibility gene 1 (BRCA1), mapped on chromosome 17q21, is implicated in the mechanisms of cellular DNA repair. Inactivation of this gene is involved in the development of many human cancers, including breast cancer. This study aimed to investigate the prognostic value of BRCA1 promoter hypermethylation and expression in breast cancer cases. Sixty-one breast cancers were examined for BRCA1 hypermethylation by methylation-specific polymerase chain reaction (PCR), and 45 paired normal breast tissues were analyzed for altered BRCA1 mRNA levels by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Aberrant methylation status in BRCA1 was detected in 15 of 61 cases (24.6%), while reduced expression was found in 7 of 45 (15.6%). BRCA1 hypermethylation was statistically associated with tumor grade III (p=0.04), a high frequency of stage IIB (p=0.02), and triple-negative phenotype (OR= 3.64, 95%CI =1.1-12.3, p=0.03). Our findings indicated that BRCA1 promoter hypermethylation is a useful prognostic marker for breast cancer.