Pectobacterium carotovorum의 검출을 위한 PCR 진단법의 개발

노지나,유미선,박동석,김정구,윤병수,No, Ji-Na,Yoo, Mi-Sun,Park, Dong-Suk,Kim, Jeong-Gu,Yoon, Byoung-Su 한국미생물학회 2009 미생물학회지 Vol.45 No.4

Pectobacterium carotovorum은 배추를 비롯한 광범위한 식물체에 무름병을 일으키는 병원성 세균으로, 무름병의 효율적인 방제를 위하여 신속한 병원체의 진단이 요구되고 있다. 따라서 본 연구에서는 높은 특이성으로 다양한 진단에 적용되고 있는 PCR법을 이용하여 Pectobacterium carotovorum을 높은 정확성과 민감성으로 검출할 수 있는 진단법을 개발하고자 하였다. 먼저 P. carotovorum에 특이성이 있다고 보고된 다양한 특이 primer들을 비교하여 가장 특이성이 높은 primer쌍들을 선별하였으며, 최종 선발된 특이 primer쌍은 ERB_3F (5'-TGCGACACCTCCTCATCACG-3'), ERB_3R (5'-CTTATCACGCTGTAACCAGC-3')로 나타났다. 이들을 사용한 PCR 검출법은 $58^{\circ}C$의 annealing 온도, 15 mM $MgCl_2$ 농도 등으로 최적화되었으며, 최적조건에서 P. carotovorum 특이 PCR 진단법은 10 pg, 즉 $2\times10^3$ copies의 병원균 유전자를 검출할 수 있는 우수한 민감성을 보였다. 또한 배양된 균주가 아닌 현장 시료에서 본 P. carotovorum 검출법을 시험해 본 결과 이 검사법은 병원균 배양없이 현장에도 직접 적용할 수 있음을 입증하였다. 따라서 본 연구를 통해 확립된 P. carotovorum 특이 PCR 진단법은 해당 병원균을 신속하게 진단하는데 유용하게 사용될 수 있을 것이라 기대한다. A new PCR method was developed to detect Pectobacterium carotovorum which is the causative agent of soft rot in Brassica pekinensis. A specific detection primer set based on Lytic murein transglycolase gene was designed and evaluated. Using ERB3_F (5'-TGC GAC ACC TCC TCA TCA CG-3') and ERB3_R (5'-CTT ATC ACG CTG TAA CCA GC-3') primers, 437 nucleotides long fragment was specifically amplified. The amplified products were observed in 52 out of 55 strains of P. carotovorum or Pectobacterium carotovorum subsp. carotovorum. On the other hand, no amplification was observed in 8 organisms including Chinese cabbage and potato. The optimal PCR condition for the ERB3_F/ERB3_R primer set was $58^{\circ}C$ for annealing and 15 mM for $MgCl_2$. With serially diluted templates, the specific PCR sensitivitie limit was $2\times10^3$ copies. Also, this method can be applied not only to DNA but also to field samples. This PCR method may be expected to be useful for specific detection of P. carotovorum.

Loop-mediated Isothermal Amplification (LAMP)법을 이용한 Chronic Bee Paralysis Virus (CBPV)의 신속 진단법 개발

노지나(Ji-Na No),유미선(Mi-Sun Yoo),Van Phu Nugyen,윤병수(Byoung Su Yoon) 한국양봉학회 2010 韓國養蜂學會誌 Vol.25 No.4

Chronic bee paralysis virus (CBPV) is the causal agent of chronic paralysis in honeybee colonies. A loop-mediated isothermal amplification (LAMP) assay allows one-step detection of gene amplification by simple reaction and requires only a simple incubator, such as water bath providing a constant temperature. In this study, CBPV-LAMP was developed for detection of CBPV. The four CBPVspecific primers named CBPV-LAMP-F3/B3/FIP/BIP were designed. CBPV was successfully amplified under isothermal conditions at about 54°C within 30 minutes and CBPV-LAMP could be detect untill 1×10⁴ copies. Especially, developed method, CBPV-LAMP was direct detection analysis by the naked eye without electrophoresis using SYBR Green I. This method provides an important diagnostic tool for the detection of CBPV infection.

IAPV내의 capsid protein gene의 검출을 위한 RT-LAMP 법의 개발

노지나(Ji-Na No),이보람(Bo-Ram Lee),유미선(Mi-Sun Yoo),윤병수(Byoung Su Yoon) 한국양봉학회 2011 韓國養蜂學會誌 Vol.26 No.2

Israel acute paralysis virus (IAPV) was reported as a virus of colony collapse disorder (CCD) in honeybee. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay allows one-step detection of gene amplification by simple analysis and requires only a simple incubator, such as water bath providing a constant temperature. RT-LAMP method was applied for simple and rapid detection of IAPV. This method was named IAPV-RT-LAMP. The four primers named IAPV-VP2-F3/B3/FIP/BIP were designed for specific amplification of VP2 capsid protein gene in IAPV. Especially, total diagnosis time was reduced until 40 miuntes including reverse transcription reaction from RNA stage. After reaction, LAMP products added fluorescent dye, SYBR Green I and GeneFinder™ were observed with naked eye under daylight without electrophoresis. IAPV-RT-LAMP is expected to be an importance diagnostic tool for detection of IAPV both in laboratory and in field.

Israeli Acute Paralysis Virus (IAPV) 검출을 위한 Semi-nested PCR 법의 개발

노지나(Ji-Na No),Nguyen Van Phu,유미선(Mi-Sun Yoo),윤병수(Byoung-Su Yoon) 한국양봉학회 2010 韓國養蜂學會誌 Vol.25 No.2

A new semi-nested PCR method was developed to detect Israeli acute paralysis virus (IAPV) which is the causative virus of colony collapse disorder (CCD) in honeybee. To amplify IAPV structural protein gene, the sequences of IAPV including accession No. ER219380, EU436455, EU436456, EU224279, EU224280, EU436423, EU218534 in GenBank were analysed. Two pairs of IAPV specific primer sets named IAPV_F1/2, IAPV_R1 outer and IAPV_F1/2, IAPV_R2 inner were designed and evaluated. As results, the unique 284bp DNA fragment was successfully amplified with high sensitivity using IAPV specific semi-nested PCR method. From only three copies of IAPV-template, the IAPV specific seminested PCR showed positive amplification. From field sample its sensitivity was also verified. The IAPV semi-nested PCR method would be applicable to the early detection of IAPV and the confirmation test for existence of few IAPVs in nature.

Loop-Mediated Isothermal Amplification (LAMP)법을 이용한 Pectobacterium carotovorum subsp. carotovorum의 신속 진단법 개발

김정구,노지나,박동석,윤병수,Kim, Jeong-Gu,No, Ji-Na,Park, Dong-Suk,Yoon, Byoung-Su 한국미생물학회 2011 미생물학회지 Vol.47 No.2

Pectobacterium carotovorum subsp. carotovorum (PCC)는 세균성 무름병균으로 주로 감자, 양배추 등의 식물에서 질병을 일으킨다. 본 연구에서는 현장에서 신속하게 진단하기 위해 loop-mediated isothermal amplification법을 이용하여 1시간 내에 등온에서 검출 가능한 진단법을 개발하였으며, 이를 PCC-LAMP법이라 명명하였다. PCC의 lytic murein transglycolase 유전자를 특이적으로 증폭시키는 4개의 프라이머를 제작하였으며 최적 온도가 $61^{\circ}C$임을 확인하고 최적 조건을 확립하였다. 최적 조건을 바탕으로 4개의 프라이머가 $1{\times}10^3$ copies까지 검출하는 민감성을 확인할 수 있었다. 본 연구에서 개발된 PCC-LAMP법은 특이성 검사를 통해 PCC만이 특이적으로 검출됨을 확인하였으며, 이는 실제 시료에서도 적용 가능함을 확인하였다. PCC-LAMP법을 통하여 PCC를 신속하고 정확하게 검출함으로써 현장에서 유용하게 적용될 수 있을 것으로 사료된다. Pectobacterium carotovorum subsp. carotovorum is the causative agent of soft rot in crops such as potato and cabbages. Loop-mediated isothermal amplification (LAMP) is a simple DNA amplification method, as well as isothermal PCR technique. In this study, a new method for the rapid detection of Pectobacterium carotovorum subsp. carotovorum was developed using LAMP that named PCC-LAMP. Based on lytic murein transglycolase gene of Pectobacterium carotovorum subsp. carotovorum, a set of four primers for LAMP was designed. The optimal PCC-LAMP reaction temperature was established at $61^{\circ}C$. Under standard conditions, PCC-LAMP amplified $1{\times}10^3$ copies of clone PCC-pBX437 per reaction. Further, this method can also assay directly by SYBR Green I without electrophoresis. Amplification was not detected for five other bacterial species. In conclusion, PCC-LAMP may be a useful method for the detection Pectobacterium carotovorum subsp. carotovorum in the field.

Loop-mediated Isothermal Amplification(LAMP) 법을 이용한 Sacbrood Virus (SBV)의 검출법 개발

이보람(Boram Lee),노지나(Ji-Na No),유미선(Mi-Sun Yoo),윤병수(Byoungsu Yoon) 한국양봉학회 2011 韓國養蜂學會誌 Vol.26 No.4

Sacbrood virus (SBV) is an infectious disease which affects the brood of honeybees, resulting in failure to pupate and death. A loop-mediated isothermal amplification (LAMP) assay allows one-step detection of gene amplification without expensive equipment such as thermocycler. The SBV-LAMP method was developed for detection of SBV easily and rapidly. A set of four designed primers named SBV-F3/B3/FIP/BIP that recognize SBV specific sequence. After optimization of conditions, SBVspecific amplification was successfully performed using SBV-LAMP from standard. SBV-specific template under isothermal conditions at 57°C within 60 minutes. The method could be detected SBV in DNA clone at 103copies/μl, also could be applied not only cDNA but also field samples. Especially, this method was developed direct detection analysis by the naked eye using SYBR Green I, Gene-FinderTM Nucleic acid fluorescent dye and phenol red. SBV-LAMP may be expected to be useful for the specific detection of SBV in field and for the monito ring of natural infection in Apis mellifera L. by SBV.

Loop-mediated Isothermal Amplification (LAMP) 법을 이용한 Nosema ceranae 검출법 개발

이보람(Boram Lee),노지나(Ji-Na No),Nguyen Van Phu,유미선(Mi-Sun Yoo),박용하(Yong-Ha Park),윤병수(Byoungsu Yoon) 한국양봉학회 2010 韓國養蜂學會誌 Vol.25 No.4

Nosema disease (Nosemosis) in honeybees is caused by obligate intracellular parasites, Nosema apis (N. apis), and also by Nosema ceranae (N. ceranae). Recently, N. ceranae was often found not only in Apis cerana, but also in European honeybees, Apis mellifera. N. ceranae was known more pathogenic and virulent to Apis mellifera than N. apis. In this study, new LAMP method for the specific detection of N. ceranae in honeybee was developed (N. ceranae-specific LAMP). Four N. ceranae-specific primers named Nosema-F3/B3/FIP/BIP were designed based on N. ceranae small subunit ribosomal RNA gene (GenBank, DQ486027). After optimization of conditions, N. ceranae-specific amplification was successfully performed using N. ceranae-specific LAMP from standard N. ceranae-specific template under isothermal conditions at 56.4℃. Also, for the application to clinical samples, N. ceranae-infected honeybees from apiaries were tested. All positive samples that were verified by Nosemaspecific real-time PCR were detected using N. ceranae-specific LAMP. N. ceranae-specific LAMP may be expected to be useful for the specific detection of N. ceranae in field and for the monitoring of natural infection in Apis mellifera by N. ceranae.

Black Queen Cell Virus 재조합 단백질의 발현

유미선(Mi-Sun Yoo),이보람(Boram Lee),노지나(Ji-Na No),Nguyen Van Phu,윤병수(Byoung-Su Yoon) 한국양봉학회 2011 韓國養蜂學會誌 Vol.26 No.4

Black queen cell virus (BQCV) causes the death of queen larvae and pupae. The coding sequence of structural protein gene in BQCV was amplified using cDNA from Apis mellifera in Korea and cloned into the pQE30-ELP (Elastin-like Polypeptide) vector at BamHI/SalI restriction enzyme site. It was presented 99% nucleotide and 100% amino acid sequence identification with BQCV sequences reported (GenBank AF183905). pQE30-BQCV-ELP was expressed in Escherichia coli M15. pQE30-BQCV in M15 was successfully expressed in the state of more than 0.1mM IPTG. This recombinant BQCV protein will be use for generation of monoclonal antibody to develop a diagnosis tool for BQCV infection in honeybee.

Loop-mediated Isothermal Amplification (LAMP) 법을 이용한 백묵병 원인균 Ascosphera apis의 검출법 개발

이보람(Boram Lee),유미선(Mi-Sun Yoo),Nguyen Van Phu,노지나(Ji-Na No),윤병수(Byoungsu Yoon) 한국양봉학회 2011 韓國養蜂學會誌 Vol.26 No.2

Chalkbrood disease of honeybee is caused by a apecies of fungus, Ascosphaera apis (A. apis), which could affect larvae of honeybee. In this study, new LAMP method for the specific detection of A. apis in honeybee was developed (A. apis-LAMP). This method is amplified DNA with high specificity, efficiency and rapidity under isothermal conditions. Four A. apis-specific primers named A. apis-F3/B3/FIP/BIP were designed based on A. apis 18S ribosomal RNA gene (GenBank, M83264.1). After optimization of conditions, A. apis-specific amplification was successfully performed using A. apis-LAMP from standard A. apis-specific template under isothermal conditions at 54℃ within 30minutes. Also, for the application to clinical samples, A. apis-infected honeybees from apiaries were tested. Visual inspection of LAMP amplifications demonstrated that the positive and negative reactions exhibit distinct and different colors in daylight, which means that gel electrophoresis is not necessary to judge the presence or absence of the A. apis. Thus, A. apis-LAMP may be expected to be useful for the specific detection of A. apis in field and for the monitoring of natural infection in Apis mellifera by A. apis.

Sacbrood Virus와 Korean Sacbrood Virus의 구별 검출을 위한 PCR법 개발

이보람(Boram Lee),Nguyen Van Phu,유미선(Mi-Sun Yoo),노지나(Ji-Na No),윤병수(Byoungsu Yoon) 한국양봉학회 2012 韓國養蜂學會誌 Vol.27 No.2

Sacbood virus (SBV) is an infectious disease, resulting in failure to pupate and death. Korean sacbrood virus (KSBV) has led to the enormous perishment of Apis cerana in Korea. In this study, PCR method was developed for the detection to distinguish SBV and KSBV using 1 primer pair. The method of discriminating detection could be easy recognized by different sizes of PCR-products, such as 318bp (SBV) and 267bp (KSBV). This primer pair was able to detect SBV until 103 copies of pBX-SBV and pBX-KSBV clone in standard assay. In addition, this method discovered the existence of KSBV in Apis mellifera. The PCR method may be expected to be useful tool for rapid, specific detection of SBV and KSBV in field and for the monitoring of natural infection in Apis mellifera and Apis cerana.