Lysiphlebus orientalis is previously recorded in 2010, which is a more recently recorded than Lysiphlebia japonica. Host range of Lysiphlebus orientalis range is narrow, while that of Lysiphlebia japonica is very broad. Although two species, Lysiphlebus orientalis and Lysiphlebia japonica, are belonged to different genus, respectively, they are morphologically similar each other, which make us confused. Therefore, we have to identify these two cryptic species using COI DNA barcode. We used the ‘NCBI-BLAST (National center for biotechnology information-Basic Local Alignment Search Tool)’ to perform COI DNA barcode identification, and introduce preliminary results in this presentation.

본 연구는 PCR 기반 RFLP (Restriction Fragment Length Polymorphism; 제한절편 길이 다형성) 분자기법을 활용하여 난 및 치어 대상 납자루아과 어류 3종의 동정을 좀 더 빠르고 정확하게 파악하고 납자루아과 어류의 종별 산란양상 및 번식생태 이해에 대한 기여가 목적이다. 본 연구를 위해 기존 선행된 문헌자료를 확인하고 납자루아과 어류가 2종 이상 동서하고 있는 지역을 확인하여 현지조사를 수행하였다. 현지조사 결과 확인된 납자루아과 어류는 묵납자루 (Acheilognathus signifer), 줄납자루 (A. yamatsutae) 및 각시붕어 (Rhodeus uyekii)로 총 3종이 확인되었으며, 확인된 납자루아과 어류와 동서하고 있는 숙주조개 (작은말조개; Unio douglasiae sinuolatus)를 채집하여 숙주조개 속 납자루아과 어류의 난 및 치어를 확보하였다. 현지조사 결과 확인된 납자루아과 어류 3종을 대상으로 미토콘드리아 DNA COI과 cyt b 유전자 염기서열을 비교하여 각각 종별로 특이성을 지닌 부위 (단일염기변이; Single Nucleotide Variation: SNV)에 맞는 제한효소를 선정하였고, 숙주조개 속 난 및 치어를 대상으로 genomic DNA를 추출하여 PCR-RFLP 실험을 수행한 결과 현지조사 시 확인된 납자루아과 어류 3종의 독특한 제한절편 길이 양상을 전기영동을 통하여 확인하였다. 본 연구를 통해 묵납자루, 줄납자루 및 각시붕어의 종을 판별할 수 있는 RFLP 마커를 개발하였으며, 숙주조개 난 및 치어를 대상으로 정확한 종의 동정을 보다 빠르고 효과적으로 수행하여 각각 납자루아과 종별 산란양상을 보다 정확히 규명하고 향후 이들 자연개체군의 효과적인 유지, 관리 및 보전 방법 개발에 유용하게 활용될 수 있을 것으로 판단된다.

The genus Aphidius is one of the largest groups of aphid parasitoids. Aphidius colemani is most common and commercially available for the aphid control. Recently, the mummies of Rhopalosiphum padi were collected from barley banker plants in Yecheon. Nucleotide sequence analysis identified its parasitoid as A. transcaspicus, which is morphologically similar to A. colemani. Interspecific variation of both COI and ITS2 between two species was 5.13 and 10.73%, respectively. Intraspecific variations of both COI and ITS2 of A. transcaspicus was 1.85 and 4.96%, respectively. We assume the barley banker plants for A. colemani were contaminated by A. transcaspicus. Thus, precise identification of natural enemies is required for more effective pest control.

Mycoplasma (M.) felis and M. canis is related with pneumonia or conjunctivitis in domestic cats and several diseases in a variety of other animals, including lower respiratory tract disease or pleuritis. Polymerase chain reaction (PCR) assays has been reported as an easy and useful method. It could be conducted even on nasal swab samples as a non-invasive rapid testing tools for large numbers of Mycoplasma species. However, PCR assays have to conduct multiple assays because of a lot of Mycoplasma species. Therefore, it need to perform several tests and reveal time consuming procedures. In this study, we developed a sensitive and specific multiplex polymerase chain reaction (PCR) assay that detects simultaneously two species like as M. felis and M. canis. The simultaneous detection of M. felis and M. canis primers were used to differentiate two mycoplasma species. The target DNA fragments were specifically amplified M. felis and M. canis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were submitted to validate the specificity of the multiplex PCR. The corresponding specific DNA products were amplified for each pathogen. The detection limit of the developed multiplex PCR is 102 pg with M. felis and M. canis DNA. Furthermore, the developed multiplex PCR detected successfully M. felis in feline nasal specimens. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. felis and M. canis in cats.

Aphidius colemani has been used as commercially available natural enemies for the aphid control. However, some siblingspecies are difficult to distinguish their morphology even though they have different parasitic characteristics. Recentlywe identified overwintering parasitoids inside the aphid mummies at the pepper greenhouse in Yecheon. The nucleotidesequence analysis identified as A. transcaspicus which is morphologically similar with A. colemani. Those genetic differencesof COI and ITS2 sequences were 4.36-4.84% and 2.65-5.84%, respectively. While A. colemani is useful for the controlof Aphis gossypii and Myzus persicae, A. transcaspicus parasitize various species belongs to the genera Hyalopterus, Melanaphisand Rhopalosiphum. Thus, precise diagnosis of natural enemies is required for the successful pest control.

The bluetongue is a serious vector-borne viral disease that affect wild and domestic ruminants. It is transmitted by midges belonging to the genus of Culicoides which count more than 1350 species. Since 1998, the disease has been spread in Europe and North Africa in 2000 including Algeria, while Korea reported serotype 1 of the virus on 2015. To know further about the existence and distribution of Culicoides species in Algeria, adult midges were collected from 17 different regions in Algeria from 2009 to 2015. The Interactive Identification Key of Culicoides (IIKC) has been used, followed by molecular identification by the sequencing of partial COI gene. At least 21 genetically different species have been identified. Among them, at least 5 potential virus vector species are present in Algeria. This study provides important insights for the understanding of genetic diversity of Culicoides and the existing potential of bluetongue spread in Algeria.

This study is the identification of root-knot nematode (RKN), Meloidogyne hapla, from strawberry in Korea using molecular analyses. Strawberry plants showed localized stunting and galled roots. Molecular analyses of COⅡ/lrRNA, 28S rDNA D2-D3 segment and internal transcribed spacer (ITS) region were employed for the identification of Meloidogyne spp. Polymerase Chain Reaction (PCR) amplification of COⅡ/lrRNA region produced a single fragment ca 528bp. Restriction digestion of the amplified PCR products with Dra1 enzyme produced two fragments at 200 and 250bp indication M. hapla. 28S rDNA D2-D3 segment and ITS were cloned and sequenced. 28S rDNA D2-D3 segment and ITS region produced a single fragment of 1004bp and 560bp, respectively. In BLAST search in Genbank, all sequences accord with other known M. hapla. As a result, all of the RKN samples were M. hapla. This study suggests that the dominant species of RKN on strawberry is M. hapla in Korea.

We developed a polymerase chain reaction (PCR)-based molecular method for sexing and identification using sexual dimorphism between the Zinc Finger-X and -Y (ZFX-ZFY) gene and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for mitochondrial DNA (mtDNA) cytochrome B (CYTB) gene in meat pieces and commercial sausages from animals of different origins. Sexual dimorphism based on the presence or absence of SINE-like sequence between ZFX and ZFY genes showed distinguishable band patterns between male and female DNA samples and were easily detected by PCR analyses. Male DNA had two PCR products appearing as distinct two bands (ZFX and ZFY), and female DNA had a single band (ZFX). Molecular identification was carried out using PCR-RFLP of CYTB gene, and showed clear species classification results. The results yielded identical information on the sexes and the species of the meat samples collected from providers without any records. The analyses for DNA isolated from commercial sausage showed that pig was the major source but several sausages originated from chicken and Atlantic cod. Applying this PCR-based molecular method was useful and yielded clear sex information and identified the species of various tissue samples originating from livestock.

본 연구에서는 국내 대표 식육인 소, 돼지, 닭, 오리의4종 식육과 염소, 양, 말, 칠면조의 4종 식육을 동시에 신속하게 감별할 수 있는 2 set의 multiplex PCR법을 개발하고자 미토콘드리아 16S RNA에서 종 특이부위를 선발하고 각 종에 대한 특이도를 높이기 위하여 인위적인 미스매치를 주어 프라이머를 제작한 후 8종 식육의 274개시료를 대상으로 특이도와 민감도를 조사하였다. 그 결과소, 돼지, 닭, 오리 모든 시료에서 각각 279, 94, 192, 477 bp의 증폭산물이, 말, 양, 염소, 칠면조의 모든 시료에서 각각 152 bp, 271 bp, 670 bp, 469 bp에서 뚜렷한 PCR 유전자 산물이 확인되어 모든 축종에서 100%의 특이도를 나타내어 축종별 감별력이 우수한 것으로 나타났다. 8종의 축종별로 DNA를 10 ng/μl으로 정량한 후 혼합물을 10배씩 단계 희석하여 반응여부를 조사한 결과, 소, 돼지, 오리에서는 100 fg까지, 닭에서는 1 pg까지 검출됨을 확인할수 있었다. 소, 돼지, 닭, 오리고기를 99.9%, 99%, 90%,70%, 50%, 30%, 10%, 1%, 0.1%의 비율로 혼합한 식육과 83℃ 20분, 100℃ 30분, 121℃ 10분에서 각각 열처리한 가열 혼합육에 대하여 검출한계를 조사한 결과 마지막단계의 희석 비율인 모든 혼합육의 0.1%에서 검출이 가능하였으며, 열처리 혼합육에서는 닭에서는 1% 농도에서소와 돼지의 혼합육에서 0.1% 농도에서 검출되어 민감도가 높음을 확인할 수 있었다. 본 연구에서 개발된 multiplex PCR법은 특이도 및 민감도에 있어서 국내 대표 식육을 감별하는데 있어서 유용한 것으로 평가된다.

Toothbrushes play an essential role in oral hygiene. However, they can be significant in microbial transmission and can increase the risk of infection, since they can serve as a reservoir for microorganisms in healthy, oral-diseased and medically ill adults. This study was conducted to evaluate toothbrush contamination in six toothbrushes donated from four people. Two participants each supplied two toothbrushes - one used in the bathroom and one used in the workplace. The other two people each donated two toothbrushes used in the workplace. Polymerase chain reaction was used to construct a 16S rRNA clone library. Sequences of cloned DNA were compared with those from the reference organisms provided by GenBank. A total 120 clones, representing 20 clones for each toothbrush, were analyzed. They are composed of six pylum, 46 genera and 79 species. The most dominant species were Streptococcus oralis, Streptococcus parasanguinis and Haemophilus parainfluenzae. Enterobacter and Escherichia were recovered from toothbrushes used domestically. Toothbrushes used in the workplace did not contain Enterobacteria.

In this study, two commercial PCR and ELISA test kits were examined for identification of eight animal species (beef, pork, chicken, duck, turkey, goat, lamb, and horse) from raw meat and meat products in Korea. The detection limit in RAW meat ELISA kit® on three types of meat samples blended with beef, pork and chicken, demonstrated that all meat species were differentiable down to 0.2%. RAW meat ELISA kit® on animal species resulted in differentiation rate of 94.5% for beef, 93.3% for pork, 90% for lamb, and 100% for chicken, duck, turkey, goat, and horse. In contrast, Powercheck Animal Species ID PCR kitTM resulted in 100% specificity at 0.05% limit of detection for all meat species. The detection limit of Cooked Meat ELISA kit® on mixed meat samples heat-treated with different temperatures and times, resulted in 0.1% for all heat-treated mixed meat except for chicken at 1.0%. Additionally, ELISA kit on sixty meat products resulted in specificity of 31.8% for ham, 13.6% for sausages, and 12.5% for ground processed products, and relatively low rate for more than 2 types of mixed meats. On the contrary, meat species differentiation using PCR kit showed higher percentage than that using ELISA kit®: 50.0% for ham, 41.7% for sausages, and 28.6% for ground processed meat. Futhermore, PCR kit on 54 dried beef meats detected pork genes in 13 products whereas ELISA kit showed negative results for all products. Hence, the possibility of cross-contamination during manufacturing process was investigated, and it was found that identical tumblers, straining trays, cutters and dryers were used in both beef and pork jerky production line, suggesting the inclusion of pork genes in beef products due to cross-contamination. In this study, PCR and ELISA test kits were found to be excellent methods for meat species differentiation in raw meat and heat-processed mixed meat. However, lower differentiation rate demonstrated in case of meat processed products raised the possibility of inclusion of other species due to cross-contamination during manufacturing process.

Precise, rapid and simple methods for species identification in animals are among the most important techniques in the livestock industry and research fields including meat classification. In this study, polymerase chain reaction (PCR) based molecular identification using inter species polymorphisms were examined by PCR-restriction fragment length polymorphism (RFLP) analysis for mitochondrial DNA (mtDNA) cytochrome b (CYTB) gene sequences among four mammalian livestock animals (cattle, horse, goat and pig). The results from PCR-RFLP analysis using the AluI restriction enzyme were also provided for the species-specific band patterns among CYTB gene sequences in these four species. The AluI-digestion for CYTB genes provided interesting migration patterns differentially displayed according to each species. Cattle and horse had one AluI-recognition site at different nucleotide positions and their AluI-digested fragments showed different band patterns on the gels. Pig had two AluI-recognition sites within the amplified CYTB sequences and produced three bands on the gels. Goat had no AluI-recognition site and was located at the same position as the uncut PCR product. The results showed the species-specific band patterns on a single gel among the four livestock animal species by AluI-RFLP. In addition, the results from blind tests for the meat samples collected from providers without any records showed the identical information on the species recorded by observing their phenotypes before slaughter. The application of this PCR-RFLP method can be useful and provide rapid, simple, and clear information regarding species identification for various tissue samples originating from tested livestock species.

Trombiculid mites are known to be the vector of tsutsugamushi disease by transmitting Orientia tsutsugamushi to human. Although the classification of trombiculid mites is necessary for vector surveillance, their classification by morphological observation is only possible at the larval stage and not easy because of similar shapes as well as tiny body sizes. Further the classification need the specimen production process, it takes much time and the accuracy of classification is changed according to the technology of the researcher. The internal transcribed spacers (ITS) regions of 8 trombiculid mite species were analyzed by amplification using tick common ITS primer sets. We designed molecular marker sets for the identification of five Leptotrombidium species, the lengths of marker L. orientale (1078 bp), L. pallidum (820 bp), L. palpale (1202 bp), L. scutellare (447 bp) and for L. zetum (621 bp) and three Neotrombicula species, the lengths of marker N. gardellai (264 bp), N. japonica (460 bp) and N. kwangneungensis (309 bp) based on alignment of ITS sequences. The markers will be helpful for exact classification of trombiculid mites. This study is the first report on molecular marker of ITS regions of trombiculid mites.

남생이(Mauremys reevesii)는 거북목 남생이과의 담수환 경에 서식하는 동물로서, 우리나라의 멸종위기야생동․식물 II급 및 천연기념물 453호로 지정되어 보호 받고 있다. 환경 부, 국립공원관리공단 및 지자체 등에서 남생이 복원 사업 이 진행되고 있지만, 남생이의 서식현황, 행동권 연구 등 기초적인 생태, 행동에 관한 연구는 일부 수행되고 있으나, 복원사업의 토대인 유전적 계통분류에 대한 연구는 미흡한 실정이다. 본 연구는 우리나라 남생이의 계통분류학적 위치 를 정립하고 복원사업의 토대를 마련하고자 국내에서 2개 지 역에서 포획된 남생이(n=2)를 대상으로 mtDNA cytochrome b 유전자 분석을 수행하였다. 남생이의 분포는 한반도, 중국 지역이며, 최근에는 대만, 홍콩, 일본, 인도네시아, 팔라우, 티모르섬에 인위적 도입으로 인한 자연 내 집단이 형성되어 있다. 일본에 서식하는 남생이는 도입종으로서 현재 일본 남 부 지방에 산재하여 서식하고 있으며, 18c 후반 한국에서 도입 된 것으로 추정되고 있다. 현재 남생이과(family Mauremys) 의 6종은 M. annamensis, M. japonica, M. mutica. M. nigricans, M. reevesii, M. sinensis이며 이들 6종간 서식처 의 유사성과 사람에 의한 인위적 도입에 따른 혼재에 의해 타종간 교배가 비교적 자유롭게 이루어지고 있어 한국에 서식하는 남생이의 유전자 분석을 통한 계통학적 위치 및 유전자 다양성의 파악이 필요하다. DNA 염기서열 분석은 많은 동물군의 계통관계 재검토 및 종과 유전자 보전을 위한 보전생물학의 연구에 중요한 자료가 되며, 미토콘드리아 DNA는 근연한 분류군이나 개 체군 내의 다양성 연구에 적합한 마커로 사용되고 있다. 본 연구에서는 한국에 서식하는 남생이의 분류학적 위치를 확 인하기 위해 cytochrome b 부분염기서열을 파악하였으며, Genbank의 염기서열과 비교하였다. 본 연구를 위해 전라남도 영암군 군서면 도갑면 도갑저수 지에서 포획된 1개체와 경상남도 산청군에서 포획된 1개체 총 2개체의 혈액 샘플에서 total DNA를 추출하였으며, PCR을 위한 primer로는 mtA, H15906을 사용하였고, PCR 조건은 40cycle, 95℃ 1min, 50℃, 72℃ 2min으로 수행하 였으며 ABI3730XL에 의해 980bp의 염기서열을 얻었다. 남생이의 타종간 교배를 확인하기 위 해 Genbank에 있는 남생이 속 내 다른 5종의 염기서열(M. annamensis (AY434564), M. japonica (AB559253), M. mutica (AY434628), M. nigricans (AJ519500), M. sinensis(AY434615))과 비교하 였다. Outgroup은 Cuora aurocapitata를 사용하였다. 염기 서열 alignment, 변이 사이트의 확인, 모델 선택, phylogenetic tree 구성을 위해 Mega5.2의 Jukes-Cantor (JC) 모델을 사 용하여 neighbor-joining tree를 만들어 비교 하였다. 남생이의 cytochrome b 960bp 결과와 Genbank에 있는 남생이 cytochrome b 960bp를 발췌해 함께 분석 한 결과 남생이 종 내의 변이 사이트는 총 13사이트 발견되었다. neighbor-joinging tree 결과 총 3clade로 나타났으며, 도갑 저수지의 1개체는 Gp1에 속하는 것으로, 경남 산청군의 1 개체는 Gp2에 속하는 것으로 확인되었다. 본 연구에서 나타난 3clade의 확인을 위해서 suzuki등이 2011에 발표한 논문과 비교한 결과 한국 서천에서 채집된 개체가 포함된 한반도 그룹에 도갑저수지의 개체가 포함되 는 것으로 나타났고, 경남 산청의 개체는 중국 그룹에 포함 되는 것으로 확인되었다. 본 연구의 도갑저수지 개체가 한 반도 그룹에 포함되어 한반도 고유종으로 판단할 수 있는 토대를 마련하였지만 샘플 수가 많지 않아 향후 한반도의 고유종에 대한 분류학적 위치를 조명하기 위해서는 남생이 샘플의 추가 확보는 물론 한국 내 권역별 샘플 확보와 더불 어 더 많은 마커의 분석이 필요할 것으로 판단된다.

본 연구에서는 팥의 꼬투리와 줄기를 가해하는 Ostrinia속 해충에 대해 종 동정 과정을 기술하였고, 사육하면서 관찰된 발육특성들을 보고 하였다. 수컷의 생식기는 3-lobed uncus 형태였으며, 가운뎃다리 종아리마디에는 털을 많이 갖는 것으로 나타났다. 미토콘드리아 cytochrome oxidase I (COI)과 II (COII) 유전자의 부분 염기서열은 콩줄기명나방(O. scapulalis), 큰섬들명나방(O. zaguliaevi), 큰조명나방(O. zealis bipatrialis)들에 대해 일본과 중국에서 보고된 서열들과 100% 일치를 보이는 종은 없었다. 기주식물 범위는 국내외 보고들 간에 일치하지 않았다. 암컷 성페로몬샘 추출물의 가스크로마토그래피 분석에서 큰섬들명나방과 큰조명나방의 성페로몬 성분인 (Z)-9-tetradecenyl acetate는 검출되지 않았다. 이상의 결과들을 종합하여 고려하였을 때, 본 연구의 팥 해충을 가해하는 곤충 종은 콩줄기명나방(O. scapulalis)일 것으로 추정되었다. 가을 야외에서 채집된 유충들을 야외조건에서 보관하였을 때, 이듬해 6월과 7월 사이에 성충들이 우화하였는데, 이 결과로부터 본 곤충 종은 말령 유충 단계에서 겨울휴면을 하는 것으로 추정되었다. 이외에 반합성 인공사료를 이용하여 실내 사육이 가능하였다.

Standardized short DNA sequences of the genome provide a DNA barcode for identifying species. Assembling DNA barcode data of regional fauna could provide a new identifying system and it will aid regional and global taxonomic studies and other applied biology. Recently, partial cytochrome c oxidase I (COI) sequences, around 670-bp region of the mitochondrial gene, has been qualified as a good DNA barcode for the identification of insect species. The superfamily Nepoidea includes two families-Belostomatidae and Nepidae. And it contains about 377 species in 23 genera worldwide. In Korea, each family is recognized 4 species 3 genera, respectively. As a part of the Korean insect DNA barcode project, we conducted preliminary DNA barcode study on Korean Nepoidea to provide useful molecular data for aiding accurate identification. This study also tested the effectiveness of a COI barcode in discriminating Korean Nepoidea species. So far, 43 individuals of 8 species belonging to two families were analyzed. We used a 658-bp fragment of COI gene was sequenced. Pairwise sequence divergences were calculated using a Kimura 2-parameter model and constructed neighbor-joining tree to calculate generic differences among species and within species.