• Korean Journal of Microbiology
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• v.43 no.4
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• pp.264-272
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• 2007

For the detection of human immunodeficiency virus (HIV), ultra-rapid real-time PCR methods were developed. The target DNA sequences were used 495 bp HIV-1-specific env gene (gi_1184090) and 294 bp HIV-2-specific env gene (gi_1332355). Ultra-rapid real-time PCR was peformed by $Genspector^{TM}$ (Samsung, Korea) using microchip-based, $6\;{\mu}l$ of reaction volume with extremely short running time in only 2 steps (denaturation, annealing/extension) in each cycle of PCR. Total reaction for 30 cycled ultra-rapid PCR detection including melting temperature analysis was completed in 7 min and 30 sec. The HIV-1-specific 117 bp-long or HIV-2-spe-cific 119 bp-long PCR products were successfully amplified from the minimum of template, $2.3{\times}10^3$ copies of each euv gene using 30 cycled two-steps ultra-rapid PCR. This kind of ultra-rapid real-time PCR method would be useful not only for the rapid-detection of HIV, but also rapid-detection of other pathogens.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.91-99
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• 2007

For the detection of Human Immunodeficiency Virus (HIV), multiple and ultra-rapid real-time PCR methods were developed. The target DNA sequences were deduced from HIV-1 specific 495bp partial env gene (gi_1184090) and from HIV-2 specific 294 bp partial env gene (gi_1332355), and were synthesized by using PCR-based gene synthesis on the reason of safety. Ultra-rapid real-time PCR was performed by $Genspector^{TM}$ using microchip-based, $1\;{\mu}l$ of reaction volume with extremely short time in each 3 step in PCR. The detection including DNA-amplification and melting temperature analysis was completed inner 15 minutes. The HIV-1 specific 117 bp-long and HIV-2 specific 119 bp-long PCR products were successfully amplified from minimum of template,2.3 molecules of each env gene. This kind of real-time PCR was designated as ultra-rapid real-time PCR in this study and it could be applied not only an alternative detection method against HIV, but also other pathogens using PCR-based detection.

• Journal of Apiculture
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• v.32 no.1
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• pp.27-39
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• 2017

PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than $10^3$ molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.

• Korean Journal of Veterinary Research
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• v.47 no.4
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• pp.399-407
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• 2007

Cause of high lethality and dissemination to human being, new development of rapid method for the detection of highly pathogenic Avian Influenza Virus (AIV) is still necessary. For the detection of AIV subtype H5N1, typical pathogenic AIV, new method to confirm sub-typing of this virus is also needed. For the purpose of ultra-rapid detection and sub-typing of hemagglutinin and neuraminidase of AIV, this study was planned. As the results we could demonstrate an ultra-rapid multiplex real-time PCR (URMRT PCR) for the detection of AIV In this study, the URMRT PCR were optimized with synthesized AIV H5- and AIV Nl-specific DNA templates and GenSpector TMC, which is a semiconductor process technology based real-time PCR system with high frequencies of temperature monitoring. Under eight minutes, the amplifications of two AIV subtype-specific PCR products were successfully and independently detected by 30 cycled ultra-rapid PCR, including melting point analysis, from $1{\times}10^3$ copies of mixed template DNA. The URMRT PCR for the detection of AIV H5N 1 developed in this study could be expected to apply not only detections of different AIVs, but also various pathogens. It was also discussed that this kind of the fastest PCR based detection method could be improved by advance of related technology in near future.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.203-211
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• 2008

Rapid detection-method for Shiga toxin type 1 that was produced from Shiga toxin-producing Escherichia coli (STEC) was developed by two-step ultra-rapid real-time (URRT) PCR. The specific primers were deduced from 80 bp stable region of stx type 1 (stxl) gene among various informations of STEC strains. URRT PCR is a microchip-based real-time PCR using 6 ${\mu}l$ of reaction volume with extremely short denaturation step and annealing/extension step (1 sec, 3 sec, respectively) in each cycle of PCR. Using the stx1-specific URRT PCR, 35 cycled PCR were finished in time of 6 min and 38 see, also measured 7 min and 28 see including melting temperature (Tm) analysis. The detection-limit of stxl-specific URRT-PCR was estimated until 3 colony forming units / PCR with products with stable Tm at $81.42{\pm}0.34^{\circ}C$. In the applications to various STEC strains and contaminated genomic DNAs, stx1-specific URRT-PCR were tested and shown that it would be expected an useful method for the rapid detection of stx1-coded STEC strains.

• Journal of Apiculture
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• v.32 no.3
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• pp.171-180
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• 2017

Slow Bee Paralysis Virus (SBPV) is a pathogenic virus against honeybee and bumblebee, causes the death of adult bee by paralyzing the fore-leg of bee. In this study, for rapid detection of SBPV from bumblebee, SBPV-specific Ultra-rapid Reverse transcription PCR was developed. After optimizing of SBPV-specific Ultra-rapid PCR, the existence of $1.0{\times}10^8$ SBPV-specific DNA molecules could be recognized in 3 minute and 35 seconds. Even $1.0{\times}10^1$ molecules of SBPV-specific DNA could be measured with quantitative manner. Meanwhile, from both imported bumblebee and bumblebee produced in Korea, SBPV were detected using proposed method. In the laboratory as well as in the field, SBPV-specific Ultra-rapid Reverse transcription PCR would be applied and might be expected as useful tools at production of bumblebee or inspection for the import and export system of bumblebee.

• Journal of Apiculture
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• v.32 no.3
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• pp.181-189
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• 2017

Lysinibacillus fusiformis has been suspected to be a pathogen of Bombus terrestris in Korea since 2008. In this study, we developed the rapid detection method for the L. fusiformis by utilizing the Ultra-rapid PCR. After optimizing of L. fusiformis-specific Ultra-rapid PCR, it can detect the existence of $1.0{\times}10^8$ L. fusiformis-specific DNA molecules in 4 minute and 22 seconds. Even, only 10 molecules could be detected quantitatively using this method. In addition, for the first time, in our knowledge, L. fusiformis was detected using proposed method from bumblebee produced commercially in Korea. Not only in the laboratory but also in the field, L. fusiformis-specific Ultra-rapid PCR would be applied and might be expected as convenient tools at production of bumblebee or inspection for the import and export of bumblebee.

• Research in Plant Disease
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• v.18 no.4
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• pp.298-303
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• 2012

Tomato yellow leaf curl virus (TYLCV), transmitted exclusively by the whitefly (Bemisia tabaci) in a circulative manner is one of the most important virus in tomato. Since the first report of TYLCV incidence in Korea in 2008, the virus has rapidly spread nationwide. TYLCV currently causes serious economic losses in tomato production in Korea. Early detection of TYLCV is one of the most important methods to allow rouging of infected tomato plants to minimize the spread of TYLCV disease. We have developed an ultra-rapid and sensitive real-time polymerase chain reaction (PCR) using a new designed real-time PCR system, GenSpectorTM TMC-1000 that is a small and portable real-time PCR machine requiring only a $5{\mu}l$ reaction volume on microchips. The new system provides ultra-high speed reaction (30 cycles in less than 15 minutes) and melting curve analysis for amplified TYLCV products. These results suggest that the short reaction time and ultra sensitivity of the GenSpector$^{TM}$-based real-time PCR technique is suitable for monitoring epidemics and pre-pandemic TYLCV disease. This is the first report for plant virus detection using an ultra-rapid real-time PCR system.

• Journal of Veterinary Science
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• v.21 no.1
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• pp.4.1-4.9
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• 2020

Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.

• Journal of Veterinary Science
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• v.22 no.3
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• pp.40.1-40.12
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• 2021

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.