• Title, Summary, Keyword: Ultra-rapid PCR

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Ultra-Rapid Two-Step Real-Time PCR for the Detection of Human Immunodeficiency Virus (HIV) (Human Immunodeficiency Virus (HIV) 검출물 위한 초고속 이단계 PCR 진단법)

  • Lee, Dong-Woo;Kim, Eul-Hwan;Yoo, Mi-Sun;Kim, Il-Uk;Yoon, Byoung-Su
    • 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.

Ultra-Rapid Real-Time PCR for the Detection of Human Immunodeficiency Virus (HIV) (Ultra Rapid Real-Time PCR에 의한 Human Immunodeficiency Virus (HIV)의 신속진단법)

  • Lee, Dong-Woo;Kim, Eul-Hwan;Yoo, Mi-Sun;Han, Sang-Hoon;Yoon, Byoung-Su
    • 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.

Development of Ultra-Rapid Multiplex PCR Detection against 6 Major Pathogens in Honeybee (꿀벌 6종 주요 병원체에 대한 초고속 다중 PCR 검출법의 개발)

  • Lim, Su-Jin;Kim, Jung-Min;Lee, Chil-Woo;Yoon, Byoung-Su
    • 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.

Development of Ultra-rapid Multiplex Real-time PCR for the Detection of Genes from Avian Influenza Virus subtype H5N1 (조류인플루엔자 H5N1 바이러스 유전자의 신속 검출을 위한 초고속 다중 실시간 PCR법의 개발)

  • Kim, Eul-Hwan;Lee, Dong-Woo;Han, Sang-Hoon;Lim, Yoon-Kyu;Yoon, Byoung-Su
    • 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.

Rapid Detection for Shiga Toxin Type 1 (Stxl) by Using Two-Step Ultra-Rapid Real-Time (URRT) PCR (초고속 이단계 PCR에 의한 Shiga 독소 타입 1의 신속 검출법)

  • Kim, Il-Wook;Kang, Min-Hee;Kwon, Soon-Hwan;Cho, Seung-Hak;Yoon, Byoung-Su
    • 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.

Rapid Detection for Lysinibacillus fusiformis, a Suspicious Pathogen of Bombus terrestris, using Ultra-Rapid PCR (초고속 유전자 증폭법을 이용한 서양뒤영벌 의심병원체 Lysinibacillus fusiformis의 신속 검출법)

  • Kim, Somin;Lim, Sujin;Kim, Jungmin;Kim, Byounghee;Tai, Truong A;Yoon, Byoungsu
    • 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.

Development of Ultra-Rapid Reverse-Transcription PCR for the Rapid Detection against Slow Bee Paralysis Virus (SBPV) (Slow Bee Paralysis Virus (SBPV) 신속 검출을 위한 초고속 역전사 중합효소 연쇄반응법의 개발)

  • Kim, Somin;Lim, Sujin;Kim, Jungmin;Lim, Yoon-Kyu;Yoon, Byoungsu
    • 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.

Ultra-rapid Real-time PCR for the Detection of Tomato yellow leaf curl virus (초고속 Real-time PCR을 이용한 Tomato yellow leaf curl virus의 신속진단)

  • Kim, Tack-Soo;Choi, Seung-Kook;Ko, Min-Jung;Lee, Minho;Choi, Hyung Seok;Lee, Se-Weon;Park, Kyungseok;Park, Jin-Woo
    • 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.

Development of Rapid Detection System for Small Hive Beetle (Aethina tumida) by using Ultra-Rapid PCR (초고속 유전자 증폭법을 이용한 벌집꼬마밑빠진벌레 (Aethina tumida)의 신속한 검출 기법 개발)

  • Kim, Jung-Min;Lim, Su-Jin;Tai, Truong A;Hong, Ki-Jeong;Yoon, Byoung-Su
    • Journal of Apiculture
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    • v.32 no.2
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    • pp.119-131
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    • 2017
  • For the Rapid detection of small hive beetle (SHB; Aethina tumida) and for the mass-survey against SHB invasion, SHB-specific ultra-rapid PCR system was developed. Three different pairs of Aethina tumida-specific primers were deduced from cytochrome oxidase subunit I (COI) gene in mitochondrial DNA of SHB. Using optimized SHB-specific ultra-rapid PCR, $2.1{\times}10^1$ molecules of COI gene belonged to SHB could be detected specifically and quantitatively within 18 minutes 40 seconds. For the purpose of the application in apiary field, a DNA extraction method from bee debris was separatedly developed. When $10^5$ SHB-specific COI molecules (1/1000 body of SHB larvae) are existed in 1g of bee debris, it could be verified inner 10 minutes as qualitative and quantitative manner. SHB-specific ultra-rapid PCR we proposed would be expected to apply widely, either in apiary field or laboratory, for the rapid detections and the control against SHB-invasion.

Development of Nested Ultra-Rapid PCR Method for the Accurate Detection of Acute Bee Paralysis Virus (ABPV) (Acute Bee Paralysis Virus (ABPV)의 정확한 검출을 위한 Nested 초고속 PCR법 개발)

  • Kim, MoonJung;Kim, JungMin;Kim, ByeongHee;Kim, SoMin;Truong, A Tai;Yoon, ByoungSu
    • Journal of Apiculture
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    • v.33 no.3
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    • pp.165-180
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    • 2018
  • Acute bee paralysis virus (ABPV) is an infectious disease causing paralysis in healthy honeybee colonies. Because ABPV is highly homologous with relative viruses, it is difficult to distinguish ABPV from especially IAPV or KBV by molecular diagnosis. In this study, for accurate and specific detection of ABPV, a secondary nested PCR detection method following the primary detection PCR against ABPV was developed. It could be able to detect from less than 100 molecules of ABPV. The detection time was also minimized from RNA extraction to PCR. Using ultra-rapid ABPV-specific detection and nested PCR, the presence of ABPV was detected from infected bee samples within 50 minutes. It might be expected that this kind of detection would be helpful for accurate detection and monitoring of ABPV in environment.