축산물 중 잔류허용기준이 설정되어 관리하고 있는 농약 azocyclotin, cyhexatin, fenbutatin oxide는 대표적인 유 기주석계 살비제이다. 기존 시험법은 가스크로마토그래피 를 사용하여 정량한계가 높고 분석 시 재현성이 떨어져 이에 대한 개선이 필요한 실정으로 본 연구에서는 비교적 간편하며 시간이 적게 소요되는 QuEChERS법을 활용하여 azocyclotin, cyhexatin, fenbutatin oxide의 시험법을 마련하 고자 하였다. 1% 아세트산을 함유한 아세트산에틸:아세토 니트릴(1:1) 혼합액을 이용하여 진탕 추출 후 d-SPE로 정 제하고 이를 농축 후 LC-MS/MS를 이용한 시험법을 개발 하였다. Azocyclotin, cyhexatin 및 fenbutatin oxide의 결정 계수(R2)는 0.99 이상으로 높은 직선성을 확인하였으며 정 량한계는 0.01 mg/kg으로 높은 감도를 나타내었다. 대표 축산물 5종(소, 돼지, 닭, 계란, 우유)에서 LOQ(0.01 mg/ kg), MRL(0.05 mg/kg), MRL 10배(0.5 mg/kg)의 농도에서 회수율 실험을 한 결과 평균 회수율이 76.4-115.3% 및 84.4-110.8%이었으며, 상대표준편차는 25.3% 이하로 나타 났다. 본 연구는 Codex 가이드라인(CAC/GL 40-1993, 2003) 및 ‘식품의약품안전처 식품의약품안전평가원의 식 품등 시험법 마련 표준절차에 관한 가이드라인(2016)’에 적합한 수준임을 확인하였다. 따라서 본 연구에서 확립한 시험법은 축산물 중 잔류할 수 있는 azocyclotin, cyhexatin, fenbutatin oxide의 안전관리를 위한 공정시험법으로 활용 가능할 것으로 판단된다.

면화는 중요한 섬유 작물로 종자는 가축의 사료로 사용 된다. 작물 생명공학은 농업 분야에서 농업적 형질과 질을 향상시키기 위해 활용되어져 왔다. 국내 식품, 사료, 가 공 제품에 유전자변형 (LM) 면화의 사용이 증가함에 따라 환경으로의 LM 면화의 비의도적 유출 또한 증가하고 있다. LMO 모니터링 사업에서 수집된 LM 면화를 검정하기 위하여 국내 수입 승인된 LM 면화의 검출법 개발이 필요하다. 본 연구에서는 LM 면화 MON757, MON88792, COT67B, GHB811 4종을 대상으로 동시검출법을 개발하였다. 이벤트에 대한 유전 정보는 유럽 JRC와 농림축산검역본부에서 확보하였다. LM 면화의 동시검출법 개발을 위해 이벤트 특 이적인 프라이머를 설계하였으며 특이적인 증폭을 확인하였다. 특이도 검정, 무작위 표준물질 혼합물 분석, 검출한계 분석을 통하여 동시검출법의 정확도와 특이도를 검증하였다. 그 결과 본 동시검출법은 각각의 이벤트를 검출할 수 있으며 LM 표준물질을 활용하여 특이도를 검정하였다. 또한 무작위 표준물질 조합도 정확하게 검출할 수 있다. 검출한 계 분석에서는 25 ng의 미량의 주형 DNA로 단회 분석으로 검출이 가능하다. 결론적으로 4종의 LM 면화 동시검출법을 개발하였으며 LM 면화 자생체 분석에 활용될 것으로 사료 된다.

A method was developed for the simultaneous detection of an antibiotic fungicide, streptomycin, and its metabolite (dihydrostreptomycin) in agricultural products using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted using methanol adjusted to pH 3 using formic acid, and purified with a HLB (Hydrophilic lipophilic balance) cartridge. The matrix-matched calibration curves were constructed using seven concentration levels, from 0.001 to 0.1 mg/kg, and linearity of five agricultural products (hulled rice, potato, soybean, mandarin, green pepper), with coefficients of determination (R2) ≥ 0.9906, for streptomycin and dihydrostreptomycin. The mean recoveries at three fortification levels (LOQ, LOQ × 10, LOQ × 50, n = 5) were from 72.0~116.5% and from 72.1~116.0%, and relative standard deviations were less than 12.3% and 12.5%, respectively. The limits of quantification (LOQ) were 0.01 mg/kg, which are satisfactory for quantification levels corresponding with the Positive List System. All optimized results satisfied the criteria ranges requested in the Codex guidelines and the Food Safety Evaluation Department guidelines. The present study could serve as a reference for the establishment of maximum residue limits and be used as basic data for detection of streptomycin and dihydrostreptomycin in food.

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.

Mycotoxins, such as aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin B1 (FMB1), ochratoxin A, T2 toxin, and zearalenone, are found in numerous vegetables. Mycotoxin accumulation in food and feed poses serious health risks to humans and animals because of carcinogenic, mutagenic, teratogenic, and toxic properties. In addition, mycotoxins cause large economic losses in commercial crop production, food and feed processing, and animal husbandry worldwide. In this study, an analytical method for the simultaneous analysis of the levels of AFB1, DON, and FMB1 in cow blood with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. AOZTM and Myco6in1TM multitoxin immunoaffinity columns and an OasisTM reversed-phase solid-phase extraction Hydrophilic-Lipophilic-Balanced columns were used to purify and concentrate the blood samples. Extracts that contained AFB1, DON, and FMB1 had average recovery of 64.0%, 98.0%, and 89.9%, respectively. In conclusion, we used LC-MS/MS to detect several important toxicological mycotoxins in cow blood. The multimycotoxin method, which detected and quantified the levels of AFB1, DON, and FMB1 can be used in animal pilot studies to monitor simultaneous exposure to major mycotoxins.

The purpose of this multiplex PCR assay is establishment and application for rapid and simultaneous detection of six pathogens related with insect diseases. Five pathogens were chosen based on the insect disease incidence rate in South Korea and specific primers of those pathogen were designed to detect insect diseases and test multiplex PCR for detecting Fungi; Beauveria bassiana(Bb), Metarhizium anisopliae(Ma), Bacteira; Bacillus thuringiensis(Bt), Pseudomonas aeruginosa(Pa), and Serratia marcescens(Sm). This research carried out the results detecting five kinds of insect pathogen of P. b. seulensis by multiplex PCR. Multiplex PCR is effective and save time to detect simultaneously these insect pathogens and multiple infections to prevent insect disease. In our study, using multiplex PCR, we demonstrated that P. b. seulensis was frequently infected with S. marcescens and co-infected with M. anisopliae in more than 80% of cases, indicating that such an analysis can be useful for pathogen identification, especially if different pathogens produce similar symptoms.

A Duplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema spp. and Vairimorpha spp. from Lepidoptera insects. Two sets of primers were selected from different genomic sequences to specifically amplify an 831 bp amplicon within the SSU rRNA gene, specific for both Nosema spp. and Vairimorpha spp. (MSSR primer); a 542 bp amplicon within the SSU rRNA gene, specific for Vairimorpha spp. (VSSU primer). Using the primers in conjunction (duplex PCR) it was possible to detect Nosema spp. and Vairimorpha spp. to differentiate between them. The sensitivity of this PCR assay was approximately 10 spores per milliliter. It is proposed that the duplex PCR is a sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting Nosema spp. and Vairimorpha spp. in Lepidoptera insect.

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.

A rapid, simple and reliable LC-MS/MS method, which can be used on a routine basis, was developed for the simultaneous detection of 8 penicillin antibiotics (amoxicillin, ampicillin, penicillin-G, penicillin-V, oxacillin, cloxacillin, nafcillin and dicloxacillin) in swine muscle and kidney. The antibiotics were extracted from samples with water and methanol. The extract was centrifuged, filtered and analyzed by liquid chromatography coupled with a tandem mass spectrometer (LC-MS/MS), using a C18 reversed phase column with water/acetonitrile gradient containing 0.05 % formic acid. Mass spectral acquisition was achieved in an electrospray positive ion mode by applying multiple reaction monitoring (MRM) of 2 fragment ion transitions to provide a high degree of sensitivity and specificity. The ion rations were consistent and could be used for confirmation of identity of the penicillin antibiotics. Recoveries of eight penicillins at three fortification levels (10, 20 and 40㎍/㎏) ranged from 79.8 to 102.4% and 72.8 to 103.4% in swine muscle and kidney, respectively. The coefficient of variation was than 9% in all samples. The estimated limits of quantification (LOQs) ranged from 1.0 to 3.2㎍/㎏ in swine muscle and kidney, respectively. The LOQs of this method are below the MRLs of penicillin antibiotics in animal tissues established in Korea and other countries.

This study was aimed to develop a novel qualitative multiplex polymerase chain reaction (PCR) for simultaneous detection of genetically modified (GM) soy and maize within a single reaction. The specific primers designed to detect four respective GM events (A2704-12, MON88017, Bt11, and MON863) were included in the tetraplex PCR system. Each of PCR products for four GM events could be distinguished by agarose gel based on their different lengths. The specificity and reproducibility of this multiplex PCR were evaluated. This multiplex PCR consistently amplified only a fragment corresponding to a specific inserted gene in each of the four GM events and also amplified all four of the PCR products in the simulated GM mixture. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM soy and maize in a single reaction.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon within SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102 spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.

본 논문에서는 식품 중 퀴놀론계(QNs) 합성항균제(marbofloxacin, norfloxacin, danofloxacin, ciprofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid 그리고 flumequine) 9종을 분석하기 위하여, 액-액 추출 과정을 거쳐서 형광검출기가 장착된 액체크로마토그래피를 이용하여 QNs를 효율적으로 동시분석하는 방법을 확립하였다. 컬럼은 Zorbax Eclipse XDB-C8(150 mm×4.6 mm, 5 μm), 이동상 용매는 200 mM ammonium acetate buffer (pH 4.5)와 ACN로 기울기 용리를 사용하였으며, 유속은 1.5 ml/min, 그리고, 주입량은 10 μl로 설정하여 분석하였다. 확립 된 분석조건으로, 표준검정 곡선은 10-500 μg/kg의 농도범위에서 상관계수가 0.9989 이상의 양호한 직선성을 나타내었으며, 회수율은 50, 100 그리고 500 μg/kg의 농도에서 89.6-106.5 %로, 향상된 추출효율을 나타내었다. 검출한계는 1-16 μg/kg이었고, 정량한계는 3-47 μg/kg이었으며, 일내(intra-day)와 일간(inter-day) 정밀도(CV%)는 0.2-15.0 %, 0.5-11.7 %이었다. 따라서, 확립된 분석방법은 광어 및 계란 중의 QNs을 효과적으로 분석하는데 이용될 수 있을 것으로 사료된다.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon with in SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.