In a previous study, Peptoniphilus mikwangii was isolated from the human oral cavity as a new species. The purpose of this study was to develop P. mikwangii-specific PCR primers. The PCR primers were designed, based on the nucleotide sequence of 16S ribosomal RNA (16S rDNA). The specificity of the primers was tested using genomic DNAs of 3 strains of P. mikwangii and 27 strains (27 species) of non-P. mikwangii bacteria. The sensitivity of primers sensitivity was determined using PCR, with serial dilutions of the purified genomic DNAs (4 ng to 4 fg) of P. mikwangii KCOM 1628T. The data showed that P. mikwangii-specific qPCR primers (B134-F11/B134-R1 & B134-F5/B134-R5) could detect only P. mikwangii strains, and 400 fg or 40 fg of P. mikwangii genome DNA. These results suggest that PCR primers are useful in detecting P. mikwangii from the oral cavity.

The purpose of this study is to develop the quantitative PCR(qPCR) assay that would enable the rapid identification and simultaneous detection of six different endodontic pathogenic bacteria in a single reaction. In this study, six pairs of primers for Treponema denticola, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Streptococcus mutans, and Staphylococcus aureus and two pairs of housekeeping genes were designed for a multiplex qPCR based on the SYBR Green method. The genomic DNA was extracted from reference strains and submitted to the qPCR reaction. The specificity of the amplified products was analyzed by melting curves. As a result, six distinct melting peaks were identified by the melting curve analysis and all of the target species were simultaneously discriminated. Therefore, the multiplex qPCR assay developed in this study can be used for rapid identification and detection of T. denticola, P. gingivalis, F. nucleatum, P. intermedia, S. mutans, and S. aureus at the same time. In combination with the melting curve analysis, the level of the target species and total bacterial load can be obtained.

인수공통 감염증의 하나인 톡소포자충의 검출을 위해서는 대부분은 ELISA 법이 사용 되고 있으나, 충체가 사멸 된 후에도 양성반응이 나타나는 등 사용에 제한이 있다. 반면 유전자 검출법은 현재 감염상태를 확인 할 수 있기 때문에 식중독 원인조사 등에 적합하다고 판단되어 이를 활용하여 본 연구를 진행하였다. 톡소포자충의 유전정보를 통해 529 repeat region의 염기서열을 얻고, 프라이머 및 TaqMan 프로브를 설계하여 real-time PCR을 이용한 검 출법을 개발하였다. 검출한계(lower limit of detection) 및 적정곡선을 확인한 결과 10 genomic DNA copy가 검출한 계로 확인되었고, 정량을 위한 곡선은 101~106 DNA copies 까지 0.999의 R2값을 나타내었다. 개발된 검출법의 증폭효 율을 비교하기 위해 B1 gene 타겟 프라이머 세트와 타입 별 검출한계를 비교한 결과, type 1, 2, 3 톡소포자충에서 같거나 더 나은 검출한계를 보였다. 또한 식품에서 주로 분리되는 식중독 세균 14종 및 원충 3종에 대해 특이도를 비교한 결과, 모두 음성으로 나타났다. 개발된 검출법을 식육검체에 적용하였을 때 type 1, 2, 3에서 모두 원활한 검출결과를 보여 증폭방해물질이 존재하지 않은 것으로 확인되었다. 본 연구를 통해 개발된 유전자검출법은 국내 유통 중인 식육에서 인수 공통감염 원충의 하나인 톡소포 자충의 감염 여부를 확인하는 사전적 모니터링의 방법으로 활용될 예정이다.

Single nucleotide polymorphisms (SNP) markers allow rapid screening of crop varieties in early growth stages. We developed a modified SNP PCR procedure for assaying SNPs in maize. For SNP marker development, we chosen 200 SNP sites from MaizeGDB database, and designed two base pair mismatch primers based on putative SNP site of B73 genome sequence. PCR products size was from 200 to 500 bp or was not shown in the case of SNP site existing in Korean silage corns. Using previously discovered 16 primer sets, we investigated distinctness of 50 silage F1 hybrid corns including 10 Korean silage corns developed by RDA such as Gangdaok, Kwangpyeongok, Dapyeongok, Andaok, Yanganok, Singwangok, Jangdaok, Cheongdaok, Pyeonggangok, and Pyeonganok as well as 40 foreign commercial silage corns. From cluster analysis, we confirmed that 10 Korean silage F1 hybrid corns were clearly distinguished except for Singwangok, P1395, and several foreign commercial corns, and selected minimum SNP primer combination for Gangdaok, Jangdaok, Pyeonggangok, and Pyeonganok. Therefore, development of SNP marker sets might be faster, cheaper, and feasible breed discrimination method through simple PCR and agarose gel electrophoresis.

Fermented food consists of a variety of lactic acid bacteria (LAB) that are also used in the industry as starter cultures. This genus comprises of different species that find importance as a preservatives in food like meat and sausages. Likewise, Lactobacillus brevis has been recognized as GRAS and is a potential probiotic strain extensively being used on an industrial scale. Since such bacteria are directly related to human health, there has been a need to identify and characterize them on the molecular level. In this study, LAB was identified and characterized from various fermented food samples available in South Korea. Two types to PCR-based molecular typing methods were used to analyze the 13 Lactobacillus brevis isolates, of which one was based on difference in the banding patterns originated on agarose gel and the other was related to the sequence analyses of various housekeeping genes in the particular strain. The former rep-PCR technique used three primers namely, REP, ERIC and (GTG)5 that amplified repetitive sequences in the genome and provided characteristic fingerprint profile for each isolate. This clustered the strains in 3 groups with the help of UPGMA method of clustering distinguishing between closely related strains. However, the latter multilocus sequencing typing (MLST) technique provided definite identification of the strains. A set of 7 housekeeping genes were determined as groEL, gyrB, rpoA, rpoB, pheS, recA and dnaK. These genes were amplified and sequenced and were subjected to comparative analysis. Discrete allelic profiles and 13 sequence types (STs) were resolved and minimum spanning tree (MST) was constructed, revealing the genetic relatedness among the isolates. On comparing the results from both the techniques, MLST proved to generate accurate and precise fingerprints owing to the sequence analysis of conserved genes thus providing a scope for research in the monitoring related species.

Monochamus alternatus (M. alternatus) and Monochamus saltuarius (M. saltuarius) are major vectors for Bursaphelenchus xylophilus in South Korea. When an adult, they are easily distinguishable by several morphological classification. However, it is difficult to identification between M. alternatus and M. saltuarius when they are larvae as they have very similar morphological characters. Thus, they are not easily distinguishable without expertise about Cerambycidae taxonomy. Furthermore, during epidemiological investigation, sometimes, adults or larvae would not be founded in death pine trees. For these reasons, in this experiment, we are able to identified between M. alternatus and M. saltuarius by mitochondrial 12S rRNA gene primers that are specific to 12S rRNA gene fragment of M. alternatus using larvae tissue and frass. Moreover, we had examined whether vectors that were already escaped from dead pine tree have Bursaphelenchus xylophilus or not by multiplex PCR using larva frass that was remained in dead pine tree.

Monochamus alternatus (M. alternatus) and Monochamus saltuarius (M. saltuarius) are major vectors for Bursaphelenchus xylophilus in South Korea. When an adult, they are easily distinguishable by several morphological classification. However, it is difficult to identification between M. alternatus and M. saltuarius when they are larvae as they have very similar morphological characters. Thus, they are not easily distinguishable without expertise about Cerambycidae taxonomy. Furthermore, during epidemiological investigation, sometimes, adults or larvae would not be founded in death pine trees. For these reasons, in this experiment, we are able to identified between M. alternatus and M. saltuarius by mitochondrial 12S rRNA gene primers that are specific to 12S rRNA gene fragment of M. alternatus using larvae tissue and frass. Moreover, we had examined whether vectors that were already escaped from dead pine tree have Bursaphelenchus xylophilus or not by multiplex PCR using larva frass that was remained in dead pine tree.

현재 제주도 감귤원에 발생하는 녹응애류는 모두 2종(Aculops pelekassi, Paracalacarus podocarpi)이며 이 중 A. pelekassi(귤녹응애, Pink citrus rust mite)는 감귤에 발생하여 감귤을 가해하고, P. podocarpi는 감귤원 주변 방풍수로 식재되는 나한송(Podocarpus macrophyllus)에 발생하여 나한송을 주로 가해한다. 하지만 녹응애는 그 크기가 0.04mm 정도여서 바람에 의해 쉽게 방풍수에서 감귤나무로 이동하여 발견되며 이를 귤녹응애로 오인하는 경우가 발생하였다. 녹응애류는 육안으로 관찰하기도 힘들뿐더러 현미경상으로도 분류 동정이 쉽지 않기 때문에 적정 방제시기 결정을 위해 PCR법을 이용한 신속하고 정확한 분류 동정법을 개발하였다. A. pelekassi와 P. podocarpi의 유전자 중 특이적인 부분을 이용하여 각각의 프라이머(332bp, 367bp)를 자체적으로 제작하였으며 이를 이용하여 A. pelekassi와 P. podocarpi 을 신속하고 정확하게 구분할 수 있었다.

두 종류의 Cronobacter 선택배지(DFI agar, R&F agar) 의 분유 및 건조호박 내 Cronobacter의 선택 분리능을 realtime PCR법과 함께 비교하였다. 분유에서의 Cronobacter 검출률은 세 가지 방법에서 유의적인 차이를 나타내지 않았으나(p < 0.05), 건조호박의 경우 R&F배지와 real-time PCR법이 DFI에서보다 유의적으로 높은 검출률을 보였다 (p < 0.05). 배지 간 선택성에 있어서도, R&F 선택배지는 건조호박에서 DFI에 비해 유의적으로 높은 선택성을 나타냈다(p < 0.05). Real-time PCR 및 R&F배지의 사용은 분유뿐만 아니라, 건조 호박 등의 높은 경쟁세균총을 갖는 영유아식의 원료로 사용될 수 있는 식품군에서도 Cronobacter를 효과적으로 검출할 수 있는 방법으로 사료된다.

Cordyceps species are important mushrooms traditionally used for heaths and vitality. C. sinensis has been widely used as a medicinal mushroom and C. militaris is popular for its substitute. C. militaris can be cultivated artificially and new strains has been improved by crossing single spore strains. As a bipolar heterothallic fungus C. militaris has two strains of compatible mating types and they can be differentiated by crossing, fruiting body formation ability and the production of perithecia. However this process is very laborious and time consuming to carry out. In this study, molecular markers were designed and used for the detection of two opposite mating types. Two mating types were assayed using two sets of primers specific for C. militaris, which were amplified a 191-bp fragment for MAT1-2 and 233-bp fragment for MAT1-1. After crossing of two compatible mating types F1 hybrids resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the multiplex PCR assays for the rapid and specific detection of both MAT1-1 and MAT1-2. This results may serve as a efficient process in the breeding program for artificial cultivation and industrial-scale production of C. militaris.

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.

칠면조는 슈퍼 푸드 중 유일한 육류 식품으로 선정되어 칠면조의 육류 소비는 더욱 증대될 것으로 기대된다. 이 에 따라 식품 중 사용된 동물성 원료인 칠면조의 신속하고 정확한 판별법 개발이 요구된다. 본 연구에서는 칠면조 를 정확하고 민감하게 검출하기 위해 TaqMan® probe를 활용한 real-time PCR 방법을 개발하였다. 종 특이 primer와 probe는 미토콘드리아의 cytochrome b 유전자를 대상으로 개발하였고, 특이성은 총 19 종의 동물성 원료를 대상으 로 확인하였다. 개발된 검출법의 검출한계는 칠면조 DNA를 10 ng 으로 부터 10배씩 희석하여 확인한 결과 0.1 pg 이었고, 동물성 식품원료인 소와 닭에 칠면조를 50, 25, 5, 1, 0.1% 비율로 혼합하여 확인한 결과 0.1 %까지 검출되 었다.

감자는 세계에서 4번째로 중요한 식량작물로 식품 및 사료로 이용되고 있다. 감자의 주요 소비형태는 일반식용 위주와 전분, 감자칩, 감자튀김 등 다양한 형태의 가공제품, 씨감자 형태로 사용한다. 또한 감자를 식품산업과 제지 산업에서 활용하기 위해 amylose 함량을 감소시키거나 acrylamide 생성을 줄인 유전자변형 감자가 개발되고 있다. 이 에 따라 우리나라의 경우 감자 가공품 수입이 많은 만큼 국내외 감자 제품에 대한 미승인 유전자변형 감자에 대한 검사법이 필요하다. International Service for the Acquisition of Agri-biotech Applications (ISAAA) 보고에 따르면 2016 년까지 45개의 GM 감자이벤트가 개발되었다. 이 중 우리나라는 식품의약품안전처로부터 4종의 GM 감자가 식품용 도로 승인되었다. 본 연구에서는 현재 미승인 유전자 감자 6개 이벤트에 대해 정성검사용 표준플라스미드 (pUC_GM potato A and B)를 제작하였으며, UDP-glucose pyrophosphorylase (UGPase) gene을 감자 내재유전자로 사용했다. 개발 된 표준플라스미드는 식품의약품안저처의 고시를 토대로 primers와 probes를 이용하여 Applied Biosystems ViiA7 real-time PCR system을 통해 정성분석을 수행하였다. 또한 검정한계치를 확인하기 위해 플라스미드를 100,000 - 10 copies까지 희석한 것을 주형으로 하여 real-time PCR을 진행하였다. 이와 같이 미승인 유전자변형 감자에 대한 정성 분석용 표준플라스미드가 개발되었으며 국내 미승인 유전자변형 감자에 대한 모니터링에 활용될 수 있을 것으로 기 대한다.

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase β-subunit gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC 33478T. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..