기후온난화 현상이 지속되고 있는 제주지역에서 환경적으로 다른 지역 모기의 계절적 발생밀도를 조사하기 위해 제주시의 국제공항, 항만 구역과 축사 그리고 서귀포 도심지의 11지점을 선정하여 3월부터 11월까지 매달 2회씩 Black light trap과 BG sentinel trap을 이용하여 모기를 채집하였다. 채집된 모기는 5속 7종. 6,042마리였으며, 이 중 빨간집모기(Culex pipiens)가 4,159마리(68.8%)로 우점종이었으며 흰줄숲모기 (Aedes albopictus)는 1,348마리(24.4%)였다. Black light trap를 이용한 채집에서 중앙동주민센터는 트랩당 72.8마리를 채집하여 모기 밀도가 가장 높게 나타났으며 제주국제공항은 트랩당 1.4마리로 가장 낮게 나타났다. BG sentinel trap을 이용한 채집에서는 항만에서 트랩당 71.7마리로 가장 많았고 도심지의 걸매생태공원에서 28.3마리로 가장 낮았다. 시기별로 모기의 밀도는 5월부터 증가하기 시작하여 8월에 1,156마리 (19.1%)로 가장 높은 밀도를 나타내었다. 채집된 암컷모기를 종별, 시기별, 지점별로 나누어 pool당 50마리 이하로 설정하여 총 364 pools에서 flavivirus 존재여부를 real time RT-PCR로 검사하였으나, 검출되지 않았다.

Potato Virus Y (PVY) (Potyviridae: potyvirus) is one of the serious emerging virus of seed potato world-wide. It affects the seed potato by transmitting non-persistently via aphids. Here, we developed a simple PVY detection method which used the boiling technique for releasing of the viral RNA from aphid such as stylet and amplification by PVY specific primers located in the viral coat protein gene which suitable for various strains. This simplified method could save the time compared to earlier detection method due to the simplified RNA extraction step. Following this procedure, we tested this one step RT-PCR based PVY detection method by using three PVY vectoring aphid species (M. persicae, A. gossypii and M. euphorbiae) as well as other sucking type insect such as thrips (F. occidentalis). This PVY detection method is rapid, easy-to-use and suitable for large-scale testing in laboratories of seed potato.

Pelargonium zonate spot virus (PZSV)는 group IV (+) ssRNA viruses, Bromoviridae에 속하는 식물 병원체로, 일반적으로 토마토, 국화, 아티초크 및 제라늄에 감염된다. 본 연구는 검역 현장에서 PZSV를 신속하고 특이적으로 진단 할 수 있는 PCR module을 개발하는 것을 목적으로 하였다. PZSV를 검출하기 위한 RT-PCR 프라이머 선발 결과, 각각 513 및 320 bp를 증폭하는2개 조합을 선발하으며, 더욱 높은 검출감도로 검출할 수 있을 뿐아니라 RT-PCR을 검증할 수 있는 nested PCR 프라이머 조합을 개발하였다. 또한, 제한효소 Xho I 부위를 삽입한 유전자변형-양성대조구 플라스미드를 설계하여, PCR module에서 대조구로부터 오염을 검증할 수 있도록 개발하였다. 본 연구에서 개발한 PCR module은 토마토, 국화, 아티초크 및 제라늄 등에서 PZSV를 간편, 신속 및 특이적으로 검출하여, 지속적으로 식물검역에 활용할 수 있을 것으로 기대된다.

PVY (Potyviridae: potyvirus) is one of the most important potato virus affecting seed potato production and also it is transmitted non-persistently via aphids. For healthy seed potato production, a virus detection system is highly important in addition to aphid monitoring and control. To achieve this detection method, it need to fast and easy to use. About two decades ago RT-PCR based PVY detection method was developed. However that was very time consuming and has low sensitivity. Here, we developed an advanced PVY detection method which a uses the boiling extraction of the viral RNA from aphid stylet and amplification by specific primers located in the viral capsid protein gene. Therefore, it could directly synthesize cDNA of PVY viral capsid gene from extracted RNA of PVY using one-step RT-PCR method in very short time compared to previous methods due to the omission of RNA extraction step. We confirmed this PVY detection method using the two aphid species (Macrosiphum euphorbiae and Aphis gossypii) that known as PVY vectors. The efficiency of this PVY detection method was 60% to 80% from two the aphid species. Hence, this method could be potentially applied to virus free seed potato production programs.

본 연구에서는 FCV 현탁액에 물리, 화학적 위생처리 후 복합효소처리라는 전처리과정을 적용한 뒤 real-time RTPCR법을 이용하여 살균효능을 분석하였다. RT-PCR 이전에 37oC에서 30분 동안 PK와 RNase A를 처리함으로써 UV, 열, 염소, 에탄올, 과초산계열 제품에 의해 불활성화 된 바이러스들은 음성 결과를 나타내었고, real-time RTPCR법을 통해 살균 효능을 정량분석한 결과, 복합효소처리를 했을 경우 무처리구보다 더 높은 살균 효능을 보이는 것을 확인할 수 있었다. 이로써 Nuanualsuwan S. 등11,18,29)의 선행연구에서와 같이 PK와 RNase A로 전처리하는 단계를 통하여 물리, 화학적 위생처리에 의해 손상되지 않은 바이러스가 RT-PCR 법에 의해 증폭되는 것을 방지함으로써 Real-time PCR법 에 대한 검출 감도를 높일 수 있음을 확인하였다. 또한, FCV를 검출하기 위해 사용된 RT-PCR과 real-time RT-PCR 두 방법 중에서도 real-time RT-PCR법이 가장 신속하면서도 민감도 높은 결과로 도출되었다. 따라서, 유전자 분석 이전에 복합효소처리는 물리, 화학적 위생처리에 의해 불활성화 된 바이러스의 RNA가 transcription 또는 증폭되는 것을 방지하기 위한 수단으로 real-time RT-PCR법과 결합 됨으로써 노로바이러스를 비롯한 식중독 바이러스를 검출 하는데 효과적으로 적용될 것으로 판단된다. 또한 식품현 장에서 전기영동 과정없이 신속하게 살아있는 바이러스만을 수치적으로 정량화함으로써 식품안전에도 기여할 것으 로 사료된다.

종자의 수입 시, 검역관련 종자전염바이러스는 가장 문제가 되는 식물병이다. 본 연구에서 PCR 검역체계가 보고되지 않은 3종의 종자전염바이러스, Cherry rasp leaf virus (CRLV), Spinach latent virus (SpLV) 및 White clover mosaic virus (WClMV)를 검출하기 위하여 reverse transcription polymerase chain reaction (RT-PCR)과 nested polymerase chain reaction (nested PCR) 방법을 도입하였다. 각각의 바이러스별로 2 세트의 RT-PCR primer가 선발되었으며, 증폭산물에서 더욱 높은 감도로 검출 할 수 있는 nested PCR primer set를 개발하였다. 본 연구에서 사용한 RT-PCR과 nested PCR 방법은 종자로부터 CRLV, SpLV 및 WClMV를 검역하는 고효율적 진단시스템으로 제공될 것이다.

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using realtime RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately 104 PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at 37oC were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

바이러스 입자를 감지하는 역전사 핵산 연쇄 증폭법 (VC/RT-PCR)은 감염된 식물종들로부터 핵산 추출 없이 식물바이러스들을 검출 할 수 있다. 본 연구는 VC/RT-PCR 분석법을이용하여 고추를 감염시키는 바이러스들을 효과적으로 진단하기 위하여 새로운 즙액 추출 완충액들이 제작하였다.토마토반점위조바이러스 (Tomato spotted wilt virus;TSWV), 고추약한모틀바이러스 (Pepper mild mottle virus;PMMoV) 및 고추모틀바이러스 (Pepper mottle virus;PepMoV) 진단을 위한 가장 최적화된 추출 완충액은 0.5%sodium sulfate를 포함하는 1.0M Tris (pH 8.0) buffer 였다.고추 바이러스들은 담배 즙액 추출 후 7일까지 검출이 되었으며, 마쇄 직후와 검출 감도는 유의한 차이가 없었다. 반면에,3가지 고추 바이러스들은 고추 즙액 추출 후 2일까지만 바이러스들이 검출되었으며, 검출 감도는 크게 감소하였다.국내 고추 재배 농가들에서 수집한 고추 시료들에서 TSWV,PMMoV, PepMoV의 단독 감염 및 PMMoV와 PepMoV의중복 감염을 선발된 최적 즙액 완충액과 VC/RT-PCR의 조합을 이용하여 동시 진단이 가능하였다.

Deformed wing virus (DWV) is a serious pathogen of the honeybee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In present study a novel micro PCR-based detection method, termed as ultra-rapid real-time PCR (UR-RT PCR), was developed for the fast and quantitative detection of DWV in honeybee. A specific detection primer set (DWV-UR-F3/R3) was used for the amplification of an unique 133-bp DNA fragment of DWV with a rapid real -time PCR system, GenSpector® TMC-1000, which proceed the cycling with fast heating and cooling rates and a small reaction volume. We showed that this method is able to detect DWV with DNA conditions, artificial recombinant DNA, pBX-DWV479 as well as with virus-infected honeybee samples. In application to a DWV-infected honey bee, the minimum detection time was 8 min 50 seconds under 30 cycles and 10min 11 seconds including melting temperature analysis. This optimizing detection method is one of the fastest real-time PCR-based diagnostic tools and is available to be applied to use for the detection in the field and of various persistency pathogens.

In the past four years, outbreaks of acute respiratory diseases associated with canine influenza H3N2 viruses in dogs and cats have been reported in South Korea and China. For prevention of disease from spread of the disease and for administration of timely medical treatments, including countermeasures for quarantine, use of a rapid and highly sensitive detection method are important to detection of the causative viruses. This study was conducted in order to develop a real time RT-PCR for the H3N2 subtype. It was based on primers targeting the highly homologous sequences of matrix, hemagglutinin, and neuraminidase genes. The detection limit of real time RT-PCR was 10 copies/ul with matrix and hemagglutinin genes, and 1 copy with neuraminidase genes, respectively. This real time RT-PCR was as sensitive as virus isolation in 52 clinical samples. The detection system developed in this study might provide more rapid and highly sensitive results than commercial rapid kits based on immunochromatographic assay.

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

dsRNA 진균 바이러스들은 효모와 버섯을 포함하여 거의 모든 종류의 곰팡이 속에서 보고되어 왔다. 자연계에 존재하 는 대부분의 진균바이러스들은 기주에 어떤 이상적인 징후 를 야기하지 않는 잠복 감염의 상태로 존재하는 반면 일부 곰 팡이 바이러스들은 기주의 기형적인 생장의 원인이 될 수 있 다고 하는데 이에 대한 연구는 특히 버섯에서 많이 이루어지 고 있다. 바이러스 병징으로 의심되는 느타리버섯 시료를 진 단용 프라이머 PVP를 이용한 RT-PCR법과 염기서열분석 으로 버섯바이러스를 검정한 결과, 비슷한 병징을 보이는 6 가지의 느타리버섯 중 3개의 시료에서 RT-PCR 특이밴드가 나타났다. 이 중 병징을 보인 느타리버섯 시료와 같은 품종을 재배하여 비교한 결과 기형 자실체와 건전 자실체 모두에서 RT-pCR 밴드를 확인할 수 있었다. 확인된 밴드를 염기서열 분석 결과 OMSV(oyster mushroom spherical virus)로 확 인되었다. 자실체의 기형과 바이러스 감염 사이에 명확한 관 련성은 없는 것으로 나타났다. 다만 PVP 바이러스이외의 다 른 바이러스에 의한 감염 가능성도 있으므로 추가적인 검정 이 필요한 것으로 사료된다.

Viruses of the honeybee, Apis mellifera L. are known to reside at low levels in colonies, typically showing no apparent signs of infection. Chronic paralysis virus(CBPV) is known to induce significant losses in honey bee colonies. The pathology is characterized by clusters of trembling, flightless, crawling bees and by individual bees, sometimes hairless, standing at the hive entrance. A minusstrand-specific RT-PCR was used to assess viral replication. This is the first report on the infection of CBPV in Korea. Using (-)RT-PCR, 27 apiaries in korea were screened for the honeybee viruses, with positive colonies being analysed for viral genetic diversity. We got 550-nt PCR product from CBPV genomic RNA. Nucleotide sequences were aligned to the complete CBPV genomic RNA sequence deposited in the GenBank database and was revealed 96%(AM-CBPV) identity, respectively. Sequence comparison with other CBPV and honeybee virus.

Metastatic spread to cervical lymph nodes(LNs) is a major determinant of outcome in oral squamous cell carcinoma (SCC). To provide an useful method for the detection of lymph node micrometastases, we fulfilled the histopathological examination and reverse transcriptase polymerase chain reaction(RT-PCR) using the paraffin-embedded LNs of oral SCC patients. In this study, 78 LNs from 12 patients with primary oral SCC were analyzed. Metastases in the regional LNs were evaluated by RT-PCR for squamous cell carcinoma antigen(SCCA) and cytokeratin 5(CK5). Detectability of metastatic LNs by RT-PCR was compared with histopathological examination. Of 78 LNs, CK5 and SCCA mRNA were detected in 32(41.0%) and 8(10.3%), respectively. Histopathologically, 10(12.8%) of 78 LNs were positive. CK5 mRNA was detected in all 10 histopathologically positive LNs. In contrast, SCCA mRNA was detected in 5 of 10 histopathologically positive LNs. These findings suggest that genetic diagnosis by RT-PCR based on CK5 mRNA expression may be sensitive and clinically useful technique to detect the presence of metastatic carcinoma cells in regional lymph nodes of oral SCC.