• Research in Plant Disease
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• v.29 no.1
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• pp.94-99
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• 2023

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

• Journal of Microbiology
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• v.40 no.1
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• pp.70-76
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• 2002

Proviral DNAs from HIV-1-infected CD4+ T cells (Molt/LAV cells) were amplified and detected in infected individual cells using polymerase chain reaction and in rifu hybridization. In this in situ PCR, three parameters were considered to achieve effective amplification and retention of amplificants inside the cells by making high molecular weight PCR products intracellularly, forming agarose matrix against the cells, and maintaining the appropriate PCR temperature profile. Over the cycles of ampliHcationl tailed primers with complementary overhanging sequences at their 5' sides manufactured high molecular weight products by using short primary products as a repeating unit. Agarose matrix could prevent the diffusion of the amplificants from the cells. Use of Thermanox coverslip inside the PCR tube offered target cells a similar temperature profile to that of conventional PCR in solution.

• International Journal of Oral Biology
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• v.41 no.3
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• pp.149-154
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• 2016

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase ${\beta}-subunit$ gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC $33478^T$. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..

• International Journal of Oral Biology
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• v.46 no.3
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• The Transactions of the Korean Institute of Electrical Engineers
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• v.41 no.2
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• pp.144-155
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• 1992

A design of a highly stable current-controlled power supply combining the phase-controlled rectifier (PCR), passive filter and active filter is investigated. A digital phase-looked voltage control (PLVC) with a capability of compensating the thyristor firing angles under unvalanced power source is proposed` otherwise the PCR output voltage has low-order subharmonics whose suppression requires a bulky passive filter. The digital PLVC has a fast dynamic characteristics as an inner control loop of the PCR. To suppress further the output ripple, an active filter using a transformer is introduced and its design is described through the frequency domain analysis. An optimal integral, proportional and measurable variable feedback (IPM) controller is designed using the time-weighted performance index based on the time domain analysis. The design method based on the time-weighted performance index gives better response characteristics than that based on the conventional performance index. It is also shown via experimental results that the proposed scheme gives better response characteristics than that based on the conventional performance index. It is also shown via experimental results that the proposed scheme gives good dynamic and static performances.

• Food Science of Animal Resources
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• v.29 no.4
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• pp.506-512
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• 2009

Salmonellosis is an important worldwide foodborne infectious disease that is transmitted by many food vehicles including raw and processed animal products and fresh produce. In this study, the effectiveness of automated ELISA ($VIDAS^{(R)}$) and realtime PCR in the detection of Salmonella spp. in steamed pork and raw broccoli sprouts was evaluated by comparing their results with those of a conventional culture method. Bulk samples (500 g) of steamed pork and raw broccoli sprouts were inoculated with various levels of Salmonella and divided into 20 samples (25 g each). All the samples, including the controls, were analyzed using a conventional culture method, $VIDAS^{(R)}$, and real-time PCR to detect the presence of Salmonella. In addition, the levels of background flora in the steamed pork and the raw broccoli sprouts were determined. In the steamed pork that contained less than 100 CFU/g of aerobic bacteria, all three methods detected low levels of Salmonella without a statistical difference in their performance. In the broccoli sprouts with high quantities of background flora (ca. $6.7{\times}10^7$ CFU/g), however, all three methods were unable to detect low levels of Salmonella, and real-time PCR and $VIDAS^{(R)}$ more sensitively detected Salmonella than the culture method, with significant statistical differences. In conclusion, $VIDAS^{(R)}$ and real-time PCR could be superior to conventional culture methods in detecting Salmonella in food with high levels of background flora.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.333-339
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• 2011

Loop-mediated isothermal amplification (LAMP) was developed to detect Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacterium (NTM) genomic DNA in blood samples of Korea native cattle. A set of four primers, two outer and two inner, were designed from M. bovis and M. avium genomic DNA targeting the IS6110 and 16S rRNA gene, respectively. Based on 85 Intradermal Tuberculin Test (ITT) positive blood sample and using conventional PCR and LAMP, the agreement quotient (kappa), which measures agreement beyond chance were 0.93 (conventional PCR) and 0.97 (LAMP), respectively. The detection limit of the LAMP method was $2.0{\times}10^2$ copy/ml M. bovis and M. avium cells, compared to $2.0{\times}10^3$ copy/ml M. bovis and M. avium cells for conventional PCR. These results suggest that the LAMP is a powerful tool for rapid, sensitive, and practical detection of MTC and NTM in blood samples of Korea native cattle.

• Genomics & Informatics
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• v.19 no.3
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• pp.30.1-30.8
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• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• International Journal of Oral Biology
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• v.40 no.4
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• pp.205-210
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• 2015

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

• International Journal of Oral Biology
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• v.44 no.3
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• pp.96-100
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• 2019

The purpose of this study was to develop Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the 16S ribosomal RNA (16S rDNA) gene. The specificity of the primers was determined by conventional PCR using 29 strains of 27 oral bacterial species including P. mikwangii. The sensitivity of the primers was determined by qPCR using the purified genomic DNA of P. mikwangii KCOM $1628^T$ (40 ng to 4 fg). The data showed that the qPCR primers (RTB134-F4/RTB134-R4) could detect P. mikwangii strains exclusively and as little as 40 fg of the genomic DNA of P. mikwangii KCOM $1628^T$. These results suggest that the developed qPCR primer pair can be useful for detecting P. mikwangii in epidemiological studies of oral bacterial infectious diseases.