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.

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.

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.

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.

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer ExpressTM software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.