• Parasites, Hosts and Diseases
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• 제24권1호
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• pp.25-41
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• 1986

뇌낭미충증환자 혈청 및 뇌척수액의 특이 IgG 항체가를 면역효소측정 법으로 측정하였을 때에 이 혈청학적 진단법이 환자진단에 얼마나 유용한지를 평가하였다. 1984년 1월부터 1986년 1월까지 주로 신경학적 중상을 나타낸 환자 355명에서 혈청 및 뇌척수액을 검사하였다. 면역효소측정법에 사용한 항원은 돼지에 자연감염된 유구낭미충의 낭액이며 단백질 농도 $2.5{\mu}g/ml$로 희석하여 사용하였고, 혈청은 1 : 100으로 희석하여, 뇌척수액은 희석하지 않고 반응시켰고 Perozidase-Conjugated Antihuman IgG goat serum(Cappel 회사제품)을 1 : 5,000으로 사용하여 혈청 및 뇌척수액내 유구낭미충 특이 IgG 항체가를 흡광도로 표시하였다. 흡광도 0.18 또는 그 이상을 양성으로 판정하였다. 그 결과를 요약하면 다음과 같다. 1. 대상자 355명중 신경외과 수술 및 병리학적 소견으로 확진된 뇌낭미충증 환자 26명, 피하결절 생검에서 낭미충증을 진단하였고 뇌전산화 단충촬영으로 확진한 환자 24명, 뇌전산화 단층환영으로 확진한 21명등 71명에서 면역효소측정법에 의한 특이 IgG항체가 양성자는 64명으로 민감도는 90.1%이었다. 그 중 혈청의 검사에 의한 민감도는 77.5%, 뇌척수액 검사에 의한 민감도는 83.1%로서 뇌척수액 검사가 더 민감한 소견이었다. 뇌낭미충증으로 확진된 환자중 위음성자는 특이 IgG 항체가가 대단히 낮은 예가 대부분이었다. 2. 대상자 355명중 뇌낭미충중 이외의 질환으로 확진된 환자는 52명으로서 그중 7례는 신경외과 수술 및 병리학적 소견에 근거하여 기타 질환으로 확진된 예이며 45례는 세균학적, 방사선학적 소견등을 근거로 기타 질환으로 확진된 예이었다. 이들 중 뇌낭미충 특이항체 검사에서 음성을 보인 예는 46례로서 이 검사의 특이도는 88.5%이었다. 혈청 및 뇌척수액검사에 의한 특이도는 카각 94.2%이었다. 위 양성반응을 보인 예 중에서 혈청 및 뇌척수액에서 모두 양성인 예는 없었다. 3. 혈청내 특이 IgG 항체 검사에 의한 기타 기생충감염자에서의 교차반응의 정도는 다음과 같았다. 무구조충증 18례중 2례, 스파르가눔증 20례중 2례, 폐흡충증 56례중 1례, 간흡충증 15례중 1례, 간질(간질)증 1례중 1례등이 교차반응을 나타내었다. 뇌척수액내 특이 IgG 함체 검사에 의한 교차반응을 뇌폐흡충증 환자 9례 중에는 없었으나 뇌스파르가눔중 환자 10례중 2례는 교차반응을 보였다. 뇌낭미충증으로 확진된 71례중 폐흡충항원에 대해 교차반응을 보인 예는 없었으나 스파르가눔 항원에 대해서는 혈청으로 검사했을 경우 6례, 뇌척수액의 경우 11례에서 교차반응을 나타내었다. 4. 뇌압상승이 있는 뇌낭미충증환자 예에서 뇌실조영술이나 뇌실복막강 단락술 도중 얻은 측뇌실 뇌척수액으로 낭미충 특이 IgG 항체가를 측정하면 측뇌실에 병변이 있지 않는 한 음성 또는 낮은 양성림위의 홉광도를 보이고 있었다. 5. 포도낭미충증 환자 4례중 혈청검사로는 4례중 3례가, 뇌척수액검사로는 검사한 3례 모두가 양성반응을 보였다. 이상의 결과는 혈청 및 뇌척수액내 특이 IgG 항체를 면역효소 측정 법으로 측정하는 혈청학적 진단법이 뇌낭미충증 환자의 감별진단에 매우 유용함을 보이고 있다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.390-394
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• 2004

The genetically modified glyphosate-tolerant soybean contains the following introduced DNA sequences: the EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene from Agrobacterium sp. strain CP4, the 35S promoter from the cauliflower mosaic virus, and the NOS terminator from Agrobacterium tumefaciens. In the present study, detection of these introduced DNAs was performed by amplification using the polymerase chain reaction (PCR). A multiplex PCR method was also applied to prevent false positive results. When primers for 35S promoter, nos3', CTP(chloroplast transit peptide), and CP4 EPSPS (EPSPS from Agrobacterium sp. CP4) were used, positive results were obtained in PCR reactions using DNA from genetically modified glyphosate-tolerant soybeans. There were no false positive results when using DNA from non-genetically modified soybeans. The CP4 EPSPS gene was detected when less than 125 pg glyphosate-tolerant soybean DNA was amplified. Lectin Lel and psb A were amplified from both non-genetically modified and genetically modified glyphosate-tolerant soybean DNA. Multiplex PCR was performed using different primer sets for actin Sacl, 35S promoter and CP4 EPSPS. The actin gene was detectable in both non-genetically modified and glyphosate-tolerant soybeans as a constant endogenous gene. Target DNAs for the 35S promoter, and CP4 EPSPS were detected in samples containing 0.01-0.1% glyphosate-tolerant soybean, although there were variations depending on primers by multiplex PCR. Soybean seeds from five plants of non-genetically modified soybean were co-cultivated for six months with those of genetically modified soybean, and they were analyzed by PCR. As a result, they were not positive for 35S promoter, nos3' or CP4 EPSPS. Therefore, these results suggest there was no natural crossing of genes between glyphosate-tolerant and non-genetically modified soybean during co-cultivation, which indicates that gene transfer between these plants is unlikely to occur in nature.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.459-468
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• 2020

This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

• 대한수의학회지
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• 제30권2호
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• pp.223-229
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• 1990

A splenectomized 5-month-old calf was inoculated with cryopreserved Theileria sergenti infected blood originated from naturally infected Korean native cattle in Chonbuk district. At peak parasitemia (40.1%), blood was collected, washed, lysed and then the T sergenti merozoite was isolated by differential centrifugation. Antigenic profile of isolated T sergenti organism was analized by SDS-PAGE and western blotting techniques. Coomassie blue stained SDS-PAGE gel revealed at least twelve protein bands of approximately 14Kd, 28Kd, 30Kd, 34Kd, 36Kd, 38Kd, 41Kd, 56Kd, 66Kd, 72Kd, 97Kd and 116Kd in the merozoite homogenate. In western blot, although T sergenti antigen recognized by specific anti-T sergenti antibodies demonstrated 28Kd, 30Kd, 38Kd, 56Kd, 58Kd, 66Kd, 97Kd and 116Kd proteins. False positive reactions were also observed in normal bovine serum with T sergenti and normal erythrocytic antigens. Therefore, predominant proteins of T sergenti merozoite antigen were found to be 28Kd, 30Kd, and 41Kd proteins of molecular weights. On going studies we will analyze the relative importance of those antigens for immunity of T sergenti in Korean native cattle.

• 한국동물위생학회지
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• 제35권4호
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• pp.269-273
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• 2012

A confirmatory serological test, the standard tube agglutination test (STAT) is evaluated for the diagnostic efficiency in brucellosis Korea. A total of 345 bovine samples were collected from regional veterinary branch under national brucellosis monitoring program from January 2010 to June 2012 in Korea. These samples were diagnosed as suspected serum and brucellosis positive by the Rose Bengal test (RBT) and the STAT, respectively. The STAT was compared and evaluated with three serological test such as the indirect-enzyme linked immunosorbent assay (I-ELISA), competitive-enzyme linked immunosorbent assay (C-ELISA) and fluorescence polarisation assay (FPA) prescribed for international trade by OIE. Among the 345 bovine serum samples, 302 (87.5%) were diagnosed as positive in the STAT, while 215 (62.3%), 223 (64.6%) and 194 (56.2%) serum samples were diagnosed as positive for brucellosis in the I-ELISA, C-ELISA and FPA, respectively. The STAT showed quite high positive results as compared with three prescribed tests of OIE. FPA, I-ELISA and C-ELISA have shown 60.6%, 64.9% and 67.2% correlation, respectively as compared to the STAT. However correlations of three prescribed tests ranged high 84.1~97.7%. Especially, correlation between I-ELISA and C-ELISA is quite high, 97.7%. These results suggest that the STAT has shown many false-positive reactions. Therefore, additional serological test, such as ELISAs and FPA, would be necessary to adopt as a confirmatory test in the national surveillance program of bovine brucellosis in Korea.

• Parasites, Hosts and Diseases
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• 제53권2호
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• 한국수산과학회지
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• 제48권2호
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• pp.158-167
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• 2015

A lateral flow immunoassay kit based on antigen-antibody interactions was developed to detect residues of beta-lactams, quinolones, tetracyclines, and sulfonamides in farmed fish. Group-specific antibodies showing cross-reactivity with other antibiotics in the same group were produced in rabbits. The rabbits were immunized eight times to obtain the maximum titers. Antibodies were extracted from the antisera collected from the immunized rabbits and produced group-specific reactions with antibiotics from the four groups. A kit was prepared that optimize conditions for the antigen-antibody reaction, using colloidal gold conjugated antibodies, and was designed to detect the four groups of antibiotics simultaneously. The kit enabled the detection of antibiotics in the four groups at below maximum residue limits (MRLs), which were $200{\mu}g/kg$ for tetracyclines, $100{\mu}g/kg$ for sulfonamides, $50{\mu}g/kg$ for beta-lactams, and $100{\mu}g/kg$ for quinolones. The cross-reactivity of the antibodies ranged from 10-80% for the sulfonamides, 20-100% for tetracyclines, 38-100% for quinolones, and 20-100% for the beta-lactams, confirming that the antibodies were group specific. The test kit was used 30 times to examine spiked antibiotics at the limits of detection (LODs) and all produced positive results, indicating high sensitivity. The LODs for the assay ranged from 4-20 ng/mL for beta-lactams, 25-50 ng/mL for sulfonamides, 20-100 ng/mL for tetracyclines, and 30-80 ng/mL for quinolones, and there were no false negative reactions at above these LODs. In addition, all of the LODs of the developed kit were correlated with high-performance liquid chromatography (HPLC) data. Our lateral flow immunoassay kit can simultaneously detect antibiotic residues from a large number of fish samples rapidly, strengthening the safety of domestic farmed and imported fish.

• 대한수의학회지
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• 제49권2호
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• pp.149-155
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• 2009

The objective of this study was to analyze data from the planned national serological monitoring program for Aujeszky's disease (AD) using a simulation model to evaluate probable outcomes expected in the sample derived from the simulated herds at predefined within-herd prevalence and herd prevalence. Additionally, prevalence at animal- and herd-level estimated by the stochastic simulation model based on the distributions of the proportion of infected herds and test-positive animals was compared with those of data from a national serological survey in 2006, in which 106,762 fattening pigs from 5,325 herds were tested for AD using a commercial ELISA kit. A fixed value of 95% was used for test sensitivity, and the specificity was modeled with a minimum, most likely and maximum of 95, 97 and 99%, respectively. The within-herd prevalence and herd prevalence was modeled using Pert and Triang distributions, respectively with a minimum, most likely and maximum point values. In all calculations, population size of 1,000 was used due to lack of representative information. The mean number of infected herds and true test-positives was estimated to be 27 herds (median = 25; 95% percentile 44) and 214 pigs (median = 196; 95% percentile 423), respectively. When testing 20 pigs (mean of 2006 survey) in each herd, there was a 3.3% probability that the potential for false-positive reactions due to less than 100% specificity of the ELISA test would be detected. It was found that the model showed prevalence of 0.21% (99% percentile 0.50%) and 0.5% (99% percentile 0.99%) at animal- and herd-level, respectively. These rates were much similar to data from the 2006 survey (0.62% versus 0.83%). The overall mean herd-level sensitivity of the 2006 survey for fattening pigs was 99.9%, with only a 0.2% probability of failing to detect at least one infected herd.

• Annals of Clinical Microbiology
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• 제22권1호
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• pp.9-13
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• 2019

배경: 다양한 임상검체에서 카바페넴분해효소 생성 장내세균(carbapenemase-producing Enterobacteriace, CPE)의 분리가 증가하고 있다. CPE 감염증은 치사율이 높고, 집단 발병의 가능성이 높아 지속적인 감시가 필수적이다. 저자들은 CPE 주요 유전자들을 한 번에 검출하기 위한 multiplex PCR을 개발하고 이를 평가하고자 하였다. 방법: 총 7종의 내성 유전자를 동시에 검출하기 위한 시발체를 새롭게 디자인하고 PCR 조건을 설정하였다. 주요 카바페넴분해효소인 KPC, IMP, VIM, NDM-1, GES, OXA-23 및 OXA-48 유전자를 대상으로 하였다. 각 유전자의 증폭 산물은 100 bp 이상의 차이가 나도록 고안하였다. 개발된 multiplex PCR은 총 69주의 CPE 양성 임상분리균주를 이용하여 평가하고, 비특이적 증폭에 의한 위양성 가능성의 배제를 위해 71주의 카바페넴 감수성 균주로 확인하였다. 결과: 본 연구에서 개발한 시발체 및 PCR 조건을 이용하여 실험한 결과 CPE 내성 유전자인 KPC 양성 14주, IMP 양성 13주, OXA-23 양성 12주, OXA-48 양성 11주, VIM 양성 9주, GES 및 NDM 양성 각 5주 등 총 69주 모두에서 CPE 유전자의 검출이 가능함을 확인하였다. 카바페넴 감수성 균주를 이용한 특이성 확인 시험에서 Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa 등을 포함한 총 71주의 다양한 그람양성 및 음성균주 모두에서 음성임을 확인하였다. 결론: 본 연구에서 개발된 multiplex PCR은 총 7종의 CPE 유전형을 한 번에 검출할 수 있어 CPE 확인 시험에 유용할 것으로 생각한다.