테트로도톡신(tetrodotoxin, TTX)은 강력한 해양생물 유 래 신경독소로, 수산물 내 TTX를 검출하기 위해 기존에 주로 사용되는 mouse bioassay (MBA)와 LC-MS/MS 기법 은 낮은 검출한계와 동물 윤리 문제 등의 한계가 있어 이 를 대체할 새로운 시험법 개발이 필요합니다. Neuro-2a assay는 대표적인 세포기반 대체 시험법으로, 이 방법은 마우스 신경모세포인 Neuro-2a 세포주에 ouabain (O)과 veratridine (V)을 처리하여 과도한 Na+ 유입으로 인한 세 포 사멸을 유도한 후, Na+ 채널 억제제인 TTX가 Na+ 유 입을 차단해 세포를 보호하는 원리를 이용해 TTX를 정량 합니다. 본 연구에서는 Neuro-2a assay를 국내 실험실 환경에 적용하기 위해 TTX 처리 조건과 O/V 농도 등의 매 개변수를 최적화하였습니다. 그 결과, 최적 O/V 농도로 600/60 μM를 설정하였으며, S자형 용량-반응 곡선이 도출 되는 8가지 농도(50-0.195 ng/mL)를 확인하였습니다. 또한, 24번의 반복 실험을 통해 데이터의 신뢰도를 평가할 수 있는 6가지 data criteria를 확립하였으며, 이 중 EC50 값 은 약 3.824-1.268 ng/mL로 나타났습니다. 실험실 간 변동 성 비교 결과, COV+와 Bottom OD값을 제외한 모든 품 질 관리 기준(quality control criteria)과 데이터 기준(data criteria)의 변동계수(CVs)는 1.31-14.92%로 도출되어, 실험 의 적정성과 재현성이 확인되었습니다. 본 연구는 국내에 서 활용 가능한 TTX 검출용 Neuro-2a assay의 최적 조 건과 신뢰성을 평가할 수 있는 quality control criteria와 data criteria를 제시하였습니다. 아울러, TTX뿐만 아니라 유사체인 4,9-anhydroTTX에 대한 TEF 값을 0.2098로 산 출하여, TTX뿐 아니라 다양한 유사체의 검출이 가능함 을 확인하였습니다. 향후, 본 시험법은 국내 수산물 내 TTX 검출을 위한 MBA 대체법으로 활용될 것으로 기대 됩니다.

마비성 패류 독소(Paralytic shellfish poisoning, PSP)는 유해 조류에 의해 생성되며, 독소에 노 출된 수산물을 섭취하였을 때 중독이 발생한다. 수산물 중 PSP를 검출하는 표준 시험법인 Mouse bioassay (MBA)는 낮은 검출한계와 동물 윤리 문제로 대체 시험법의 개발 필요성이 대 두되고 있다. 이러한 대체 시험법 중, PSP가 신경 세포막의 Na+ 채널을 차단하는 기전을 이 용한 마우스 뇌신경 모세포종 세포 기반 시험법(Neuro-2a assay)의 표준화를 위한 노력이 대두 되고 있다. Neuro-2a assay의 원리는 Neuro-2a 세포주에 Na+/K+ ATPase 억제제인 Ouabain (O)과 Na+ 채널 활성화제인 Veratridine (V)을 처리하여 과도한 Na+ 유입으로 인한 세포사멸을 유도한 상태에서, Na+ 채널 억제제인 PSP를 처리하게 되면 Na+ 유입이 차단되어 세포가 생존 하는 것을 측정하는 것이다. 본 연구에서는 PSP 검출을 위한 Neuro-2a assay를 국내 연구 환 경에 맞게 다양한 매개변수를 개선하여 최적 시험법을 확립하고자 하였다. 고려한 매개변수 들은 세포밀도, 배양 조건 및 PSP 처리 조건 등으로, 그 결과는 아래와 같다. 초기 세포밀도 는 40,000 cells/well로, 세포 배양시간 및 처리시간은 각각 24시간으로 설정하였다. 또한 최적 O/V 농도는 500/50 μM로 설정하였다. 본 연구에서 PSP 중 Saxitoxin (STX)에 대해서 O/V 처 리가 된 상태에서 S자형 용량-반응 그래프가 도출되는 8가지 농도(368~47,056 fg/μl)를 확인 하였고, Neuro-2a assay의 실험실 간 변동성 비교를 통해, 실험의 적정성 확인을 위한 5가지 Quality Control Criteria와 실험 데이터의 신뢰가능 범위(Data Criteria) 6가지를 설정하였다. 확 립된 조건으로 Neuro-2a assay를 진행한 결과 반수영향농도(EC50) 값은 약 1,800~3,500 fg/μl 로 나타났다. 실험실 간 변동성 비교 결과, Quality Control Criteria 값 및 Data criteria 값의 변 동계수(coefficients of variation (CVs))가 1.98~29.15% 범위로 산출되어 실험의 적정성 및 재현 성이 확인되었다. 본 연구를 통해 우리나라에서 활용할 수 있는 PSP 검출용 Neuro-2a assay 시험법의 최적 조건 및 5가지 Quality control 기준을 제시하였고, PSP 중 대표적인 독소인 STX 을 대상으로 Neuro-2a assay를 실시한 결과 유의한 EC50 값을 산출할 수 있었으며, 향후 국 내 수산물을 대상으로 MBA를 대체할 수 있는 PSP 검출법으로 활용될 것으로 기대된다.

Spodoptera eridania and S. ornithogalli (Lepidoptera: Noctuidae), which are polyphagous pests that damage various crops such as tomatoes and beans are regulated quarantine species that are highly likely to invade South Korea. Therefore, it is crucial to promptly and accurately identify the presence of S. eridania and S. ornithogalli in crop fields to effectively eradicate as a regulated quarantine species. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay, which allows for rapid in-field identification. To develop the LAMP　assay, we selected target species-specific genomic regions from the whole-genome sequences of one target and 13 other lepidopteran species. We validated each five and six primer sets that consistently produced positive reactions in S. eridania and S. ornithogalli, respectively. To test the sensitivity of the each locus, LAMP reactions were performed using various reaction times using crude DNA, which was extracted from various types of adult tissues. All sensitivity tests were also successful.

토복령은 우수한 항균, 항산화, 항염증 효능을 가진 소재로 알려져 있다. 이러한 토복령(Smilax china)의 추출물의 기능성을 화장품에 적용하기 위한 기초연구로써 토복령에서 발견되는 플라보노이드인 quercetin, catechin, naringenin의 농도별 경피 투과 특성을 조사할 필요성이 있다. Marzulli의 정의에 적 용한 결과 케르세틴의 Kp 값은 0.1 mg/mL에서 "빠름"으로 분류되었고, 0.2 및 0.4 mg/mL에서 "보통"으로 분류되었다. 특히, 농도가 증가함에 따라 투과 속도가 감소하는 경향이 있었다. 나린제닌의 경우 Flux 값은 각각 0.1, 0.2 및 0.4 mg/mL 농도에서 0.69, 1.07 및 1.42 μg/hr/cm²이었으며, 해당 Kp 값은 각각 6.95, 5.34 및 3.56이었다. 나린제닌의 Kp 값은 모든 농도에서 "보통" 범주에 속하며, 케르세틴과 관찰된 것과 같이 농도가 높아짐에 따라 투과 속도가 감소하였다. 카테킨의 경우 Flux 값은 각각 0.1, 0.2 및 0.4 mg/mL 농도에서 0.75, 1.09 및 1.66 μg/hr/cm²이었으며, 해당 Kp 값은 각각 7.55, 5.46 및 4.16이었다. 카테킨의 Kp 값은 모든 농도에서 일관되게 "보통"으로 분류되었다. 여드름 저해능 및 항염증 효능이 우수 한 토복령 추출물의 유효성분인 quercetin, catechin, naringenin의 경피 투과 특성이 보통 이상으로 나타나 기능성 화장품에 사용할 수 있는 우수한 천연물 소재인 것을 확인할 수 있었다.

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), which is native to tropical and subtropical regions of the Western Hemisphere is now annually arrives in Korea. In this study, we developed loop-mediated isothermal amplification (LAMP) assay, one of the main merits of which is a rapid identification of target species. Five among 11 FAW-specific loci tested successfully provided a consistent reaction when ten FAWs, which were collected from eight localities in four countries were tested, whereas the 13 non-target species were not amplified. To increase in-field applicability of the method all life stages, reaction time, and different periods after death was tested using the quick extracted DNA. Our FAW diagnostic protocol can be completed within 30 min, from the process of extracting genomic DNA from an egg or a 1st instar larva to species determination.

In order to permanently dispose of radioactive waste drums generated from nuclear power plants, disposal suitability must be demonstrated and the nuclides and radioactivity contained in the waste drums, including those in the shielding drums, must be identified. At present, reliable measurements of the nuclide concentration are performed using drum nuclide analysis devices at power plants and disposal facilities during acceptance inspection. The essential functions required to perform nuclide analysis using the non-destructive assay system are the correction for self-attenuation and the dead time correction. Until now, measurements have mainly been performed for drums containing solid waste such as DAW drums using SGS calibration drums with ordinary iron drums. However, for drums containing non-uniform radioactive waste, such as waste filters embedded in cement within shielding drums, a separate calibration drum needs to be produced. In order to produce calibration drums for shielded and embedded waste drums, the design considered the placement of calibration sources, setting of shielding thickness, correction for medium density, and cement mixing ratio. Based on these considerations, three calibration drums were produced. First, a shielding drum with an empty interior was produced. Second, a density correction drum filled with cement was produced to create apparent density on the surface of the shielding drum. Third, a physical model drum was produced containing a mock waste filter and cement filled in the shielding drum.

This study was conducted to obtain basic information for the use of the ATP fluorescence detection method in consideration of the most common and frequent contamination situation that occurs in laboratories dealing with fire blight causing bacterium, Erwinia amylovora. ATP luminescence measurements (Relative Light Unit, RLU) were tested against these pathogen cells (CFU/cm2) which were artificially introduced on the disinfected surface of a bench floor of a biosafety cabinet (Class 2 Type A1), on a part of the disinfected surface of a lab experimental bench, on a part of the disinfected floor, and on a part of the disinfected floor of an acryl chamber for bioaerosol studies in a biosafety laboratory (BSL 2 class) using two different ATP bioluminometers. RLU values were not much increased with the bacterial cells from 2.15 × 102/cm2 to 2.15 × 106/cm2. RLU values varied among the four different surfaces tested. RLU values measured from the same number of bacterial cells differed little between the two different ATP bioluminometers used for this study. RLU values obtained from bacterial cells higher than 2.15 × 107/cm2 indicated the presence of bacterial contamination on the four different surfaces tested. The R2 values obtained based on the correlation data for the RLU values in response to different E. amylovora cell numbers (CFU/ cm2) on the surfaces of the four test spots ranged from 0.9827 to 0.9999.

Fescues, which are widely cultivated as grasses and forages around the world, are often naturally infected with the endophyte, Epichloë. This fungus, transmitted through seeds, imparts resistance to drying and herbivorous insects in its host without causing any external damage, thereby contributing to the adaptation of the host to the environment and maintaining a symbiosis. However, some endophytes, such as E. coenophialum synthesize ergovaline or lolitrem B, which accumulate in the plant and impart anti-mammalian properties. For example, when livestock consume excessive amounts of grass containing toxic endophytes, problems associated with neuromuscular abnormalities, such as convulsions, paralysis, high fever, decreased milk production, reproductive disorders, and even death, can occur. Therefore, pre-inoculation with non-toxic endogenous fungi or management with endophyte-free grass is important in preventing damage to livestock and producing high-quality forage. To date, the diagnosis of endophytes has been mainly performed by observation under a microscope following staining, or by performing an immune blot assay using a monoclonal antibody. Recently, the polymerase chain reaction (PCR)-based molecular diagnostic method is gaining importance in the fields of agriculture, livestock, and healthcare given the method’s advantages. These include faster results, with greater accuracy and sensitivity than those obtained using conventional diagnostic methods. For the diagnosis of endophytes, the nested PCR method is the only available option developed; however, it is limited by the fact that the level of toxic alkaloid synthesis cannot be estimated. Therefore, in this study, we aimed to develop a triplex real-time PCR diagnostic method that can determine the presence or absence of endophyte infection using DNA extracted from seeds within 1 h, while simultaneously detecting easD and LtmC genes, which are related to toxic alkaloid synthesis. This new method was then also applied to real field samples.

The ovary undergoes substantial physiological changes along with estrus phase to mediate negative/positive feedback to the upstream reproductive tissues and to play a role in producing a fertilizable oocyte in the developing follicles. However, the disorder of estrus cycle in female can lead to diseases, such as cystic ovary which is directly associated with decline of overall reproductive performance. In gene expression studies of ovaries, quantitative reverse transcription polymerase chain reaction (qPCR) assay has been widely applied. During this assay, although normalization of target genes against reference genes (RGs) has been indispensably conducted, the expression of RGs is also variable in each experimental condition which can result in false conclusion. Because the understanding for stable RG in porcine ovaries was still limited, we attempted to assess the stability of RGs from the pool of ten commonly used RGs (18S, B2M, PPIA, RPL4, SDHA, ACTB, GAPDH, HPRT1, YWHAZ, and TBP) in the porcine ovaries under different estrus phase (follicular and luteal phase) and cystic condition, using stable RG-finding programs (geNorm, Normfinder, and BestKeeper). The significant (p < 0.01) differences in Ct values of RGs in the porcine ovaries under different conditions were identified. In assessing the stability of RGs, three programs comprehensively agreed that TBP and YWHAZ were suitable RGs to study porcine ovaries under different conditions but ACTB and GAPDH were inappropriate RGs in this experimental condition. We hope that these results contribute to plan the experiment design in the field of reproductive physiology in pigs as reference data.

In this study, newly improved Ferron assay test haved on timed spectrometry was used for the determination of hyolrolytic Al species presented in PACl coagulant. The color development reagent ferron was prepared by using conventional method and two newly developed methods. Then the ferron assay test was used to compare and analyze the distribution of Al(III) hydrolyzed species presented in the prepared PACl and alum. The preparing method of reagent A required an aging period of 7 days by adding a hydroxylamine hydroxide and a 1,10-phenanthroline monohydrate reagent, whereas the preparing method of reagent B was used as a coloring agent immediately without aging time. The regression analysis between UV absorbance and Al concentrations of conventional method and newly developed method of ferron reagents in low-concentration aluminum solutions and high-concentration aluminum solutions, showed the correlation coefficients of 0.999 or higher, as showing high correlations of conventional method and newly developed method. Applying Ferron assay test, Al species in the PACls and alum were classified as Ala(monomeric Al), Alb (polymeric Al), and Alc (colloidal and precipitated Al). Distribution of Al(III) hydrolyzed species according to the preparation of ferron colorimetric reagents was similar.