The swine is one of the most widespread mammalian throughout the whole world. Presently, many studies concer-ning microsatellites in swine, especially domestic pigs, have been carried out in order to investigate general diversity patterns among either populations or breeds. Until now, a lot of time and effort spend into a single PCR method. But simple and more rapid multiplex PCR methods have been developed. The purpose of this study is to develop a robust set of microsatellites markers (MS marker) for traceability and individual identification. Using multiplex-PCR method with 23 MS marker divided 2 set, various alleles occurring to 5 swine breed (Berkshire, Landrace, Yorkshire, Duroc and Korea native pig) used markers to determine allele frequency and heterozygosity. MS marker found 4 alle-les at SW403, S0227, SWR414, SW1041 and SW1377. The most were found 10 alleles at SW1920. Heterozygosity represented the lowest value of 0.102 at SWR414 and highest value of 0.861 at SW1920. So, it was recognized appro-priate allele frequency for individual identification in swine. Using multiplex-PCR method, MS markers used to determine individual identification biomarker and breed-specific marker for faster, more accurate and lower analysis cost. Based on this result, a scientific basis was established to the existing pedigree data by applying genetics additio-nally. Swine traceability is expected to be very useful system and be conducted nationwide in future.

The chigger mite, Leptotrombidium pallidum, is widely distributed throughout South Korea and is a major vector for Orientia tsutsugamushi, the causative agent of scrub typhus. In this study, the genome size of the chigger mite was estimated to determine the necessary coverage level prior to whole genome sequencing. Cloning of EF1α and RpS3 as putative single copy reference genes were conducted and their partial sequences were determined. Using the serially diluted reference genes with known amount as standard templates, the weight of a single copy of the genome was predicted by a method based on quantitative real time PCR. The average genome length estimated from the weight using two methods was 191 ± 7 Mb. When the genome size of other arthropods (Drosophila melanogster, Apis mellifera and Tetranychus urticae), with their genome analysis completed, were estimated using the same method and compared with actual values, the estimation accuracy was 79.8-98.9%, suggesting our current estimation of L. pallidum genome size is reliable. The estimated L. pallidum genome size is in a similar range to other Acariform mites, such as the dust mite and scabie mite, but appoximately 10-fold smaller compared to the deer tick, which belongs to Parasitiform. Our finding provides key information for further genome sequencing and understanding of mite genome evolution.

Virus infections of the honeybee(Apis mellifera) have been increasingly investigated during the last decade. In general, honeybee viruses are widespread and most of them persist as inapparent infections. We screened honeybee colonies for the presence of several bee viruses, including deformed wing virus(DWV), black queen virus(BQCV), Kashmir bee virus(KBV), Israeli acute paralysis virus (IAPV), sacbrood virus(SBV), acute bee paralysis virus(ABPV), using uniplex RT-PCR. Frequently simultaneous infections with different viruses are diagnosed in seemingly healthy bee colonies. Therefore we developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses.

Sacbrood virus (SBV) is one of the most destructive honey bee virus. The virus causes failure to pupate and kills honey bee larvae. The infacted larvae`s color is change to brown. At the end, honey bee colony is destructed. Recently Korean Scabrood virus(KSBV) caused a great loss of Korean honey bee(Apis cerena) colonies for short period. Therefore, We need a highly rapid diagnosis method for rapid detection of KSBV. In this study, We need amicro-scale chip-based real-time PCR system (GeneChecker®). This system was developed for rapid, specific PCR based diagnosis. This system has uncommonly fast heating and cooling system. So We was able to detecting of KSBV in Apis cerena in short time. This system needs small reaction volume(total 10ul). This volume include SsoFast™ Evagreen Supermix and serially diluted cDNA templates showed a high sensitivity of 101copies.That machine can setting each PCR stage time. A specific detection primer set (KSBV-123-F/R) was used to amplify a unique 123bp DNA fragment. This PCR assays using serially diluted cDNA templates showed a high sensitivity of 101 copies. When applied to KSBV-positve samples, the result showed high specifity. The minimum diagnosis time was 9m 47s (30cycle). The amplied positive samples appear red fluorescent color. This novel detection method could be used a PCR-based diagnositic tool (GeneChecker®). The results showed high sensitivity and specifity in short time. And this diagnosis method is expected to be applied to rapidly detect various pathogens.

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.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema Spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema Spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA(SSU rRNA) gene of N. ceranae was successfully amplified and sequenced among examined genes, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR(qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentrations as low as 0.85 ng/μl genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators. Recently, pathogens and parasites affect the life span and fecundity of their host and been isolated from B. terristris. In order to detect viral infection in the field populations of B. terristris, we collected adults and isolated total RNA for reverse transcriptase-polymerase chain reaction (PCR). The PCR primers specific for several viruses such as deformed wing virus, Israel acute paralysis virus, Kashmir bee virus and black queen cell virus (BQCV) were newly designed and applied to gene amplification for cloning. Only BQCV was successfully amplified and sequenced, which suggests that BQCV may mainly infects the examined field population of B. terristris. To detect of capsid protein gene of BQCV, 4 selected regions were analyzed by primary PCR and 1 region was successfully amplified, which was further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that BQCV was detected at concentrations as low as 0.1ng/μl total RNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terristris.

농업적, 환경적, 경제적 및 사회적인 이익으로 농업생명공학에 의한 유전자변형(GM) 작물의 재배는 점차 증가되고 있다.국내에서도 주요 작물을 대상으로 유용 GM작물이 개발되고있으며, 최근 Choline kinase 유전자(OsCK1)가 도입된 병저항성 형질전환벼가 개발되었다. GMO의 안전성과 관련하여, 표시제의 시행 또는 사후 이력추적을 위해서 검정법이 필수적으로 요구되고 있다. 본 연구에서 각 134, 306, 243bp의 PCR증폭산물을 갖는 유전자 특이, 구조 특이 및 이벤트 특이primer를 병저항성(OsCK1) GM벼의 검출에 사용하였고, 다른어떤 작물에서도 반응산물을 나타내지 않았다. 이벤트 특이primer CKRB32-1/02-2를 사용한 정성 duplex PCR을 통해서OsCK1 GM벼에 대한 검출한계(LOD)가 0.05%임이 확인되었다. Real-time PCR을 이용한 정량검정을 위해서 벼 내재유전자 염기와 OsCK1 GM벼의 5’-인접염기를 갖는 pSPSCKR을표준물질로 제조하였고, 10 copies 범위까지 정량검출이 가능한 것으로 나타났다. 따라서 도출된 real-time PCR 방법의 정확성 및 정밀성을 확인하고자 0.5, 1, 3, 5 및 10%로 GM시료에 대하여 정량 분석하였으며, 표준편차 및 상대표준변이가 20% 내로 확인되었다. 이상의 결과로, 개발된 이벤트 특이정성 및 정량 PCR 방법이 OsCK1 GM벼의 사후 GMO 모니터링 및 이력추적에 효과적으로 적용 가능할 것으로 판단된다.

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

Chrysanthemum stunt viroid (CSVd) is a serious pathogen affecting chrysanthemum that has caused significant economic losses to Chrysanthemum flower production worldwide. Control of CSVd disease is difficult due to its contagious nature and long latent period in the field. As chrysanthemum is most often produced by implanting seedlings, it is necessary to diagnose CSVd infection before cultivation. In this study, we screened CSVd infection in seedlings from 30 varieties including 5 domestic, 6 Japanese, and 19 European varieties. Molecular diagnosis of the combination of RT-PCR and nested PCR showed that CSVd was not detected by the first RT-PCR but detected by the second nested PCR analysis in 10 varieties, including 1 domestic, 2 Japanese, and 7 European varieties. Further comparison of 10 identified CSVd nucleotide sequences showed that those are highly conserved (99-100%) and the most similar to an isolate (AB006737) identified in Hokkaido, Japan. Our study suggests that the combination of RT-PCR and nested PCR analysis is successful for the CSVd diagnosis of seedlings and the molecular diagnosis is necessary to prevent the introduction and propagation of viroid disease into the fields.

Real Time PCR을 이용한 벼 흰잎마름병균의 밀도를 측정할 수 있는 새로운 방법을 개발하였다. Real Time PCR을 이용하여 벼 흰잎마름병을 예찰하기 위하여 TaqMan MGB probe를 이용한 프라이머인 Xan_PahgeF & Xan_PahgeR primer와 Xan_Pahge FAM MGB probe를 제작하였으며, 높은 특이성이 인정되었다. 병원균 배양액, 벼 흰잎마름병원균의 DNA, 병원세균에 오염된 물에서도 효과적으로 검출이 되었다. 농수로물이나 관개수에서 벼 흰잎마름병균의 밀도를 측정할 때 정량 PCR의 저해인자 제거 후 신속하게 벼 흰잎마름병균의 밀도를 측정할 수 있다.

본 연구에서는 분자생물학적 방법을 통한 알레르기 유발 원재료 확인을 위해 PCR방법의 최적 조건을 구축하였 다. 가공식품에서 알레르기 원료성분 확인을 위하여 200bp 내외의 PCR 산물을 생성할 수 있는 종특이 프라이머를 설계하거나 선행연구사업 결과를 활용하였다. 대상 원료로는 국내 식품알레르기 표시대상인 난류, 우유, 메밀, 땅 콩, 대두, 밀, 고등어, 게, 새우, 돼지고기, 복숭아 및 토마 토와 제외국에서 알레르기 유발 성분으로 규정하고있는 아몬드, 참깨를 포함하여 총 14종을 대상으로 하였다. 특이 프라이머를 사용하여 PCR 한 결과 난류, 우유, 메밀, 땅콩, 대두, 밀, 고등어, 게, 새우, 돼지고기, 복숭아, 토마토, 아몬드 및 참깨로부터 각각 281, 131, 138, 120, 118, 127, 211, 174, 231, 138, 174, 132, 103 및 220bp의 특이 밴드를 확인하였으며 상호간의 비특이적 밴드는 검출되 지 않았다. 본 연구에서 확립한 알레르기 유발 원재료 검출법은 식품의 부정확한 표시나 가공식품의 제조과정 중 알레르기 유발물질의 비의도적 혼입 등으로부터 소비자를 보호하고 향후 수출 제품에 있어서 정확한 알레르기 유발 원재료 표기에 활용이 가능할 것으로 판단된다.

우리나라와 전 세계의 여러 지역에서 수집한 비늘버섯속 18 균주와 개암비늘버섯 2 균주를 대상으로rDNA의 ITS region 염기서열과 genomic DNA의RAPD-PCR을 수행하였다. ITS1과 ITS2영역의 염기의수는 각각 233~271, 158~233 그리고 174~219 염기쌍으로 종에 따라 변이가 있었는데 ITS2영역의 염기서열이 ITS1의 영역보다 변이가 높았고 5.8S지역의 염기의 수는 비교적 변이가 적었다. 각각의 균주 간 유연관계를 알아보기 위해 ITS영역의 염기서열을 이용하여계통도를 작성한 결과 실험에 사용한 균주는 8개의 클러스터로 나누어지는 것으로 나타났으며 동일한 종의버섯은 동일한 클러스터에 속하는 것으로 나타났다.또한 20종류의 primer를 이용하여 비늘버섯속 버섯을대상으로 RAPD-PCR을 수행한 결과 15개의 primer가효과적으로 염색체 DNA를 증폭하는 것으로 나타났다.증폭의 양상은 primer의 종류와 종에 따라 변이가 있었다. 이 결과를 토대로 계통수를 작성한 결과 계통수는 ITS 영역의 PCR 결과와 매우 유사하였다. 본 실험결과, 실험에 사용한 비늘버섯속 버섯의 종과 계통 간의 유연관계는 높았으며, rDNA ITS 영역의 염기서열분석 결과를 이용해 공시된 각각의 비늘버섯 종을 분류하는데 유용하게 사용이 가능하였다.

The objective of this study was to develop PCR primers that are specific for Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii. We designed the S. sanguinis-, S. parasanguinis-, and S. gordonii- specific primers, Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 respectively, based on the nucleotide sequences of the Ssa21, Spa17, and Sgo41 DNA probes that were screened using inverted dot blot hybridization (IDBH). The species-specificity of these primers was as- sessed against 43 strains of mitis group streptococci, in- cluding clinical strains of S. sanguinis, S. parasanguinis, and S. gordonii. The resulting PCR data revealed that species-specific amplicons had been obtained from all strains of the target species tested, and that none of these amp- licons occurred in any other strains from other species. These results suggest that the Ssa21-F3/Ssa21-R2, Spa17- F/Spa17-R, and Sgo41-F1/Sgo41-R1 primers may be useful in detecting S. sanguinis, S. parasanguinis, and S. gordonii at the species level, respectively.

Bombus terrestris has played an important role in the pollination in agricultural fields for the alternatives in colony collapsing in the honeybee. Recently, some pathogens or parasites such as viruses, bacteria, mites have been discovered in B. terristris, which affects its life span and fecundity. In order to detect a microsporidian, Nosema apis. in the field population, we collected honeybees and isolated genomic DNA. PCR primers specific for 16S ribosomal RNA (16S rRNA) were synthesized and applied to gene amplification for cloning and quantitative real-time PCR (qRT-PCR). The amplified gene was cloned and sequenced to confirm the 16S rRNA gene. qRT-PCR analysis showed the detection limit of 16S rRNA of Nosema apis was approximately 0.5 ng/μl genomic DNA. This result suggests that detection via qRT-PCR can be applied for the diagnosis of pathogen infection.

벼멸구(Nilaparvata lugens), 흰등멸구(Sogatella furcifera), 애멸구(Laodelphax striatellus)는 우리나라 벼 재배에서 가장 중요한 멸구류 해충이다. 야외 포장에서 3 종 멸구의 어린 약충은 전문가들도 육안으로 구분이 어려운 실정이며, 매년 비래량 을 조사하기 위해 유아등을 통해 채집되는 벼멸구 성충의 경우 기타멸구들과 상당 히 유사한 형태를 가지고 있기 때문에 육안으로 구분하기가 쉽지 않다. 따라서 벼멸 구 비래량에 대한 정확한 자료 수집과 효율적인 방제 전략 수립을 위해서는 육안 이 외의 보조적인 방법으로 벼멸구, 흰등멸구, 애멸구와 기타멸구류를 정확하게 동정 해 내는 과정이 필요하다. 본 연구는 3대 멸구류의 종 특이적 프라이머 선발을 위해 수행되었고 동정 시간 및 비용 절감을 위해 Direct PCR 방법을 선택하였다. 예상 종 특이 프라이머들은 COI, COII, microsatellite 유전자에 기반하여 총 23개를 제작 검토하였다. 실험 결과, 벼멸구는 BPH_5를 사용하여 340bp 근처에서, 흰등멸구는 WBPH_03와 WBPH_04를 사용하여 각각 160bp와 170bp 근처에서, 애멸구는 WBPH_656을 사용하여 510bp 근처에서 band 형성을 확인하였다. 첫 번째 혼합 프 라이머 세트(BPH_5+WBPH_03+WBPH_656)와 두 번째 혼합 프라이머 세트 (BPH_5+WBPH_04+WBPH_656) 모두 벼멸구, 흰등멸구, 애멸구 DNA에 대해 band 형성 및 구분이 가능하였고, 기타멸구(북방멸구, 일본멸구, 들판멸구, 벼멸구 붙이, 겨풀멸구)에서는 어떠한 band 형성도 발견할 수 없었다. 다만 첫 번째 프라이 머 세트와는 달리 두 번째 혼합 프라이머 세트는 3종 모두 200bp에서 공통적인 band를 나타내었고, 이는 PCR시 정상적인 증폭 유무를 확인하는 척도가 될수 있을 것으로 판단되어 두 번째 혼합 프라이머 세트가 멸구류 3종 동정에 보다 유용하리 라고 생각된다.

콩과(Fabaceae) 작물 해충인 팥나방(Matsumuraeses phaseoli)과 어리팥나방(M. falcana) (나비목: 잎말이나방과)은 형태적으로 매우 유사하여 종 구별이 힘든 것으로 알려져 있다. 본 연구에서는 PCR-SSP(PCR with Sequence Specific Primers) 방법으로 두 종을 빠르고 정확하게 구별할 수 있는 판별법을 찾고자 두 종의 미토콘드리아 시토크롬 옥시다제 I(mtCOI) DNA 부분영역(439 bp)의 염기서열을 해독하였다. 그리고 다른 나방 종의 mtCOI 염기서열과 함께 나열하여 비교한 후 팥나방과 어리팥나방에서 종 특이적으로 차이가 나는 단일 뉴클레오티드를 프라이머의 3ʹ말단으로 하는 염기서열 특이 프라이머 조합을 만들었다. PCR 산물들을 전기영동 한 결과, 어리팥나방은 245 bp, 팥나방은 409 bp와 245 bp의 특이적 밴드 패턴을 보여 두 종을 구별할 수 있었다.

Lepidopteran pests monitoring in adult stage was generally performed using delta or corn typed trap including rubber septa impregnated sex pheromone (lure). Sometimes, unfortunately trapped samples were severly damaged because of biotic and/or abiotic environments such as micro-organism, predator and rain, sticky material, respectively. In our case, we monitored potato tuber moth, PTM, Phthorimaea operculella distribution during 2009~2012 in Korea. However, we encountered unexpected problem, another species can be trapped in species specific sex pheromone trap. Therefore, species confirmation was needed in trapped samples. Here we developed confirmation method by direct PCR (without DNA extraction) or sequencing methods which trapped samples that cannot identified by morphologically. We designed multi-plex PCR universal primers and species specific primers in rRNA region because to check the success of PCR and species identification. This direct PCR method can be applied in other species confirmation which monitored using pheromone trap.

In order to determine an authenticity of food ingredient, we used DNA barcode method by universal primers. For identification of animal food ingredients, LCO1490/HCO2198 and VF2/FISH R2 designed for amplifying cytochrome c oxidase subunit1 (CO1) region and L14724/H15915 for cytochrome b (cyt b) region on mitochondrial DNA were used. Livestock (cow, pig, goat, sheep, a horse and deer) was amplified by LCO1490/HCO 2198, VF2/FISH R2 and L14724/H15915 primers. Poultry (chicken, duck, turkey and ostrich) was amplified by LCO1490/HCO 2198 and VF2/FISH R2 primers. But, Fishes (walleye pollack, herring, codfish, blue codfish, trout, tuna and rockfish) were only amplified by VF2/FISH R2 primers. For plant food ingredients, 3 types of primers (trnH/ psbA, rpoB 1F/4R and rbcL 1F/724R) have been used an intergenic spacer, a RNA polymerase beta subunit and a ribulose bisphosphate carboxylase region on plastid, respectively. Garlic, onion, radish, green tea and spinach were amplified by trnH/psbA, rpoB 1F/4R and rbcL 1F/724R. The PCR product sizes were same by rpoB 1F/4R and rbcL 1F/724R but, the PCR product size using trnH/psbA primer was different with others for plants each. We established PCR condition and universal primer selection for 17 item's raw materials for foods and determine base sequences aim to PCR products in this study. This study can apply to determine an authenticity of foods through making an comparison between databases and base sequences in gene bank. Therefore, DNA barcode method using universal primers can be a useful for species identification techniques not only raw materials but also processed foods that are difficult to analyze by chemical analysis.