• The Plant Pathology Journal
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• v.40 no.4
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• pp.408-413
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• 2024

The emergence of rice black-streaked dwarf virus (RBSDV) poses a significant threat to global cereal crop cultivation, necessitating the urgent development of reliable detection and quantification techniques. This study introduces a reliable approach for the precise and sensitive quantification of the RBSDV in cereal crop samples, employing a reverse transcription digital polymerase chain reaction (RT-dPCR) assay. We assessed the specificity and sensitivity of the RT-dPCR assay proposed for precise RBSDV detection and quantification. Our findings demonstrate that RT-dPCR was specific for detection of RBSDV, with no cross-reactivity observed with other viruses infecting cereal crops. The RT-dPCR sensitivity was over 10 times that of RT-quantitative PCR (RT-qPCR). The detection limit of RT-dPCR was 0.096 copies/㎕. In addition, evaluation of RT-dPCR assay with field samples was conducted on 60 different cereal crop samples revealed that RT-dPCR (45/60) exhibited superior accuracy compared with RT-qPCR (23/60). In this study, we present a specific and accurate RT-dPCR assay for the detection and quantification of RBSDV.

• Korean Journal of Ichthyology
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• v.19 no.2
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• pp.88-92
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• 2007

The effect of dexamethasone injection on the burden of marine birnavirus (MABV) in asymptomatically infected olive flounder (Paralichthys olivaceus) fingerlings was investigated. In real time PCR analysis, the threshold cycle (Ct) value of the fish injected with dexamethasone was significantly lower than that of the fish in the PBS-injected and no-handling groups. The higher amplification of the MABV gene in the dexamethasone-injected group than the 2 control groups was confirmed also by semi-quantitative RT-PCR. The results indicate an increase of MABV burden in olive flounder fingerlings after a single injection with dexamethasone.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.205-211
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• 2006

A quantitative analytical method to detect new lines of genetically modified (GM) maize, NK603 and TC1507, has been developed by using a real-time polymerase chain reaction (PCR). To detect these GM lines, two specific primer pairs and probes were designed. A plasmid as a reference molecule was constructed from an endogenous DNA sequence of maize, a universal sequence of a cauliflower mosaic virus (CaMV) 35S promoter used in most GMOs, and each DNA sequence specific to the NK603 and TC1507 lines. For the validation of this method, the test samples of 0, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% each of the NK603 and TC1507 GM maize were quantitated. At the 3.0% level, the biases (mean vs. true value) for the NK603 and TC1507 lines were 3.3% and 15.7%, respectively, and their relative standard deviations were 7.2% and 5.5%, respectively. These results indicate that the PCR method developed in this study can be used to quantitatively detect the NK603 and TC1507 lines of GM maize.

• Journal of Microbiology and Biotechnology
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• v.22 no.10
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• pp.1350-1358
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• 2012

Viral pathogens, alongside other pathogens, have major effects on crustacean aquaculture. Hepatopancreatic parvovirus (HPV) is an emerging virus in the shrimp industry and has been detected in shrimp farms worldwide. The HPV genome has greater diversity than other shrimp viruses owing to its wide host range and geographical distribution. Therefore, developing diagnostic tools is essential to detect even small copy numbers from the target region of native HPV isolates. We have developed two easy to use quantitative real-time PCR kits, called Green Star and Dual Star, which contain all of the necessary components for real-time PCR, including HPV primers, using the primers obtained from the sequences of HPV isolates from Korea, and analyzed their specificity, efficiency, and reproducibility. These two kits could detect from 1 to $1{\times}10^9$ copies of cloned HPV DNA. The minimum detection limits obtained from HPV-infected shrimp were $7.74{\times}10^1$ and $9.06{\times}10^1$ copies in the Green Star and Dual Star assay kits, respectively. These kits can be used for rapid, sensitive, and efficient screening for HPV isolates from Korea before the introduction of postlarval stages into culture ponds, thereby decreasing the incidence of early development of the disease.

• International Journal of Oral Biology
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• v.46 no.1
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• pp.1-6
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• 2021

Since the outbreak of coronavirus disease 2019 (COVID-2019), the infection has spread worldwide due to the highly contagious nature of severe acute syndrome coronavirus (SARS-CoV-2). To manage SARS-CoV-2, the development of diagnostic assays that can quickly and accurately identify the disease in patients is necessary. Currently, nucleic acid-based testing and serology-based testing are two widely used approaches. Of these, nucleic acid-based testing with quantitative reverse transcription-PCR (RT-qPCR) using nasopharyngeal (NP) and/or oropharyngeal (OP) swabs is considered to be the gold standard. Recently, the use of saliva samples has been considered as an alternative method of sample collection. Compared to the NP and OP swab methods, saliva specimens have several advantages. Saliva specimens are easier to collect. Self-collection of saliva specimens can reduce the risk of infection to healthcare providers and reduce sample collection time and cost. Until recently, the sensitivity and accuracy of the data obtained using saliva specimens for SARS-CoV-2 detection was controversial. However, recent clinical research has found that sensitive and reliable data can be obtained from saliva specimens using RT-qPCR, with approximately 81% to 95% correspondence with the data obtained from NP and OP swabs. These data suggest that self-collected saliva is an alternative option for the diagnosis of COVID-19.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.12
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• pp.1665-1671
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• 2013

Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.4
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• pp.275-283
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• 2007

Objective: This study was performed to investigate the expressions of endometriosis related genes in shed endometrial tissues from menstrual blood of patients with or without endometriosis. Methods: The shed endometrial tissues were collected on 2$^{nd}$ or 3$^{rd}$ day of menstrual cycle with Wallace catheter in patients with endometriosis (n=16) and without endometriosis (n=26). The mRNA expressions of twelve kinds of endometriosis related genes were compared between two groups using semi-quantitative RT-PCR. Results: The collected shed endometrium was confirmed by histological observation. Expressions of telomerase, c-kit and aromatase mRNA were not detected by RT-PCR in shed endometrial tissues. The mRNA expressions of apoptosis related genes (fas, fas ligand, bcl-2, bax), stem cell factor, estrogen receptor-$\alpha$/$\alpha$, endometriosis protein-I and secretory leukocyte protease inhibitor gene were similar between shed endometrial tissues with endometriosis and without endometriosis. Conclusion: We could not find the difference of mRNA expressions of tested endometriosis related genes between shed endometrial tissues with or without endometriosis by semi-quantitative RT-PCR analysis. It may be related to the dynamical changes of gene expressions in the endometrium with menstrual cycle.

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1026-1033
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• 2014

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

• 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.

• 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'.