• 대한의생명과학회지
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• 제21권1호
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• pp.9-14
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• 2015

Mouse hepatitis virus (MHV) is a major pathogen in laboratory mice that usually leads to fatal diseases, such as hepatitis, multiple sclerosis, encephalitis, and respiratory disease. MHV has a high infection rate, and it needs to be detected as soon as possible to prevent its spread to other facilities. However, MHV detection by enzyme-linked immunosorbent assay (ELISA) often gives false positives; thus, it is very important that the results are confirmed as true positives in the early infection stage or distinguished as false positives with more accurate, reliable methods. Under microbiological screening, MHV ELISA-positive mice were found in four GFP-tagging transgenic mice. To verify the detection of the MHV antigen directly, reverse transcription polymerase chain reaction (RT-PCR) was performed, and the mice were determined to be MHV negative. Additional serum antibody-based screening was conducted with three different ELISA kits, and multiplexed fluorometric immunoassay (MFIA) was performed to confirm their accuracy/sensitivity. In brief, the ELISA kit for A59 nucleocapsid protein (MHV-A59N) revealed MHV ELISA positivity, while other ELISA kits (MHV-S lysate and MHV-JHM lysate) demonstrated MHV negativity. In MFIA, only the test for the recombinant A59 nucleocapsid antigen was MHV positive, which was consistent with the ELISA results. These results suggest that the ELISA kit with the recombinant A59 nucleocapsid antigen might induce non-specific MHV ELISA positivity and that confirmation is therefore essential.

• 한국축산식품학회지
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• 제31권5호
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• pp.658-662
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• 2011

In this study, 543 samples of press hams, sausages, processed ground meat and processed cheese acquired from retail markets in Seoul and Gyeonggi province in Korea from 2005 to 2010 were monitored using a one-step multiplex polymerase chain reaction (PCR) method that involves the amplification of specific soya or maize endogenous genes and the amplification of 35S promoter (p35S) and nopaline synthase terminator (tNOS) for GMO detection. Among the 543 samples, 477 samples were amplified for maize and/or soybean endogenous genes. Although one sausage sample collected in 2008 showed amplification of tNOS, the result was assumed to be false positive based on the results from further tests of other sausage samples of the same brand. Our results demonstrate the absence of GM soya and/or maze of livestock products in the Korean market during 2005-2010. In addition, the one-step multiplex PCR using previously constructed primer sets appears to be useful as a screening method for the detection of GMOs in processed livestock products. However, more specific methods should be established and employed to detect the event-specific GM gene for positive reaction samples by screening tests in processed livestock products.

• Animal Bioscience
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• 제34권12호
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• pp.1995-2002
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• 2021

Objective: Detection of adulteration in processed meats is an important issue for some countries due to substitution of beef with a cheaper source of protein like poultry. In this study, the presence of chicken meat was investigated using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to verify adulteration of fermented sausage samples. Methods: A total of 60 commercial samples were collected from 20 establishments in three replicates including 10 fermented sausage manufacturers and 10 butchers to investigate the presence of chicken meat with the sequential use of real-time PCR and ELISA techniques. In addition, pH, moisture content, water activity and color values of the samples were determined. Results: Both real-time PCR and ELISA showed agreement on the presence or absence of chicken meat in 55 out of 60 fermented sausage samples and chicken meat was identified with both methods in 16 samples. Five samples produced inconsistent results for the presence of chicken meat in the first run. Nevertheless, the presence of chicken meat was verified with both methods when these samples were analyzed for the second time. In addition, the average physico-chemical values of the fermented sausage samples tested positive for chicken meat were not significantly different from some of those fermented sausage samples tested negative for the chicken meat. Conclusion: The sequential use of real-time PCR and ELISA techniques in fermented sausages could be beneficial for the government testing programs to eliminate false negatives for detection of adulteration with chicken meat. Furthermore, consumers should not rely on some of the quality cues including color to predict the adulteration of fermented sausages with chicken meat since there were no statistical differences among some of the samples tested positive and negative for chicken meat.

• 대한의생명과학회지
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• 제27권1호
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• pp.35-43
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• 2021

Human Sapovirus (HuSaV) is one of the major causes of acute gastroenteritis in humans, and it is used as a molecular diagnostic technique based on polymerase chain reaction (PCR) from humans, food, shellfish, and aquatic environments. In this study, the HuSaV diagnosis technique was used in an aquatic environment where a number of PCR inhibitors are included and pathogens, such as viruses, are estimated to exist at low concentration levels. HuSaV-specific primers are improved to detect 38 strains registered in the National Center for Biotechnology Information (NCBI). The established optimal condition and the composition, including the RT-nested PCR primers and SL® Non-specific reaction inhibitor, were found to have 100 times higher sensitivity based on HuSaV plasmid than the previously reported methods (100 ag based on HuSaV plasmid 1 ng/μL). Through an artificial infection test, the developed method was able to detect at least 1 fg/μL of HuSaV plasmid contaminated with total nucleic acid extracted from groundwater. In addition, RT-nested PCR primer sets for HuSaV detection can react, and a positive control is developed to verify false positives. This study is expected to be used as a HuSaV monitoring method in the future and applied to the safety response to HuSaV from water environments.

• 대한임상검사과학회지
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• 제41권1호
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• pp.42-46
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• 2009

Unlike most bacteria, Treponema pallidum subspecies cannot be readily isolated or sustained in cell culture for numerous generations. In korea, two non treponemal tests are currently considered as standard; the VDRL slide test and RPR card test. These tests are based on an antigen composed of an alcoholic solution containing measured amount of cardiolipin, cholesterol, and sufficient purified lecithin to produce reactivity. The nontreponemal reagin tests measure immunoglobulin M (IgM) and IgG to lipoidal material released from damaged host cells as well as to lipoprotein-like material and possibly by cardiolipin released from the treponemes. The object of the evaluation was to evaluate the performance of the Mediace RPR kit on the automated biochemistry analyzer system as a method for screen method of syphilis as well as to identify BFP possibility. For evaluation of routine screening test, a total 2,380 specimens tested by Mediace RPR from 28th Oct, 2007 to 22th Feb, 2008. For evaluation of BFP possiblility, we measured samples which have potential BFP reaction in Syphilis test such as ANA (anti-nuclear antibody) positive (135 samples), CRP (C-reactive protein) positive (100 samples), RF (Rheumatoid factor) positive (26 samples), and other potential BFP cases (17 samples) including total 278 samples. These samples were tested quantitative test Mediace RPR with Hitachi 7600 P module. For comparison with current manual test, VDRL slide test were performed. Of these 2380 specimens, 2350 were negative, 30 were positive, and one were positive with TPHA. Both methods agreed for 2356 (98.9%) samples. Of the 30 samples showed positive results over 1.0 R.U, 6 samples showed positive results with VDRL test. Of these 6 samples, 1 samples showed positive with TPHA test. The combination of the Automated Biochemistry analyzer and VDRL test for retest can be increase efficiency of syphilis screening test.

• 한국수정란이식학회지
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• 제10권2호
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• pp.145-151
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• 1995

초기배의 성판정은 대상가축의 성을 선발하는 수단으로써 가축의 육종 및 번식에 있어 가치가 매우 높다. 체세포, 체외수정 또는 처녀발생 초기배의 성을 결정하기 위해 capillary polymerase chain reaction (PCR)을 이용하였으며 성판정에 이용되는 상실배 또는 배반포는 체외수정과 그 후의 난관상피세포와의 공배양에 의해 생산되었다. 초기배의 genomic DNA는 0.2$\mu$g/$\mu$L proteinase K를 함유하고 있는 PCR lysis buffer에 하나의 초기배를 부유하게 50˚C 에서 1시간동안 배양한 후 95˚C에서 10분간 효소를 비활성화시킴으로써 준비되었다. 웅성 초기배에서는 두개의 증폭산물(소특이)만이 생산되었다. 이 기법에 의한 성판정 결과 초기배의 성비는 예상되는 1:1의 성비와 유의적인 차이가 없었다. 잔여 난구세포 또는 투명대에 결합된 정자 등으로 인한 잘못된 성판정이 종종 발생하였는데, 이는 citrate 처리후 투명대를 완전히 제거함으로써 false positive 또는 negative 결과를 극복할 수 있었다. 이상과 같은 결과는 체외생산 소 초기배의 신속하고(2시간 증폭) 정확한 성판정의 가능성과 초기배 이외의 세포로부터의 오염이 확립된 수세방법에 의해 효과적으로 배제될 수 있음을 제시하였다.

• 대한치과의사협회지
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• 제15권8호
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• pp.611-615
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• 1977

The author observed histochemically the nature of the calcified tissue in the rabbit dental pulp, induced by pulpal injection of potassium permanganate. The pulp of rabbit mandibular incisors were exposed and enlarged by a dental hand reamer. The exposed pulps were injected with 0.05ml of 20mM solution of potassium permanganate dissolved in Ringer's solution in experimental tooth. Also the control tooth received a pulpal injection of 0.05ml of Ringer's solution. After pulpal injection, the tooth was plugged with a gutta-percha root canal point. The staining techniques were hematoxylin-eosin stain, van Gieson stain, PAS reaction, toluidine blue stain, alcian blue-hematoxylin stain and colloidal iron-picric acid stain. The results were as follows: 1. The pulp on experimental tooth showed osteodentin-like calcified tissue. Also, in some areas, false denticle-like substance were observed. 2. The central portion of the calcified matrix showed metachromasia in toluidine blue stain had strong staining capacity in alcian blue stain. 3. The peripheral portion of the calcified tissue revealed marked van Gieson positive reaction for collagen. But their staining ability in alcian blue was slight and metachromasia was not appeared.

• Parasites, Hosts and Diseases
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• 제60권4호
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• pp.295-299
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• 2022

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

• International Journal of Oral Biology
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• 제44권1호
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• pp.8-13
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• 2019

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to $3{\mu}M$ or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, $2{\mu}M$ of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

• 한국콘텐츠학회논문지
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• 제21권1호
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• pp.465-474
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• 2021

'심각한 급성 호흡기 증후군 코로나 바이러스 2(SARS-CoV-2)'에 의한 질병인 코로나-19는 2020년 3월 세계 보건기구에서 세계적인 전염병 대유행으로 선언되었고, 대부분의 나라에서 선별 및 확진을 위한 진단검사법으로 실시간 중합효소 연쇄반응 검사를 시행한다. 그러나 국가별 목표유전자 및 프로토콜이 다를 뿐만 아니라 진단결과의 판독절차도 다양해서 국가별로 확진자의 기준 역시 다르다. 이에 본 종설에서는 세계보건기구에서 고시한 국가별 목표유전자 및 검사기법, 진단기준을 비교하였고, 검사의 특이도와 민감도, 최소검출 한계, 양성 및 음성 대조군, 교차반응 후보군, 검체 대조군 설정 등의 특이사항도 함께 살펴보았다. 또한 각국의 검사기법과 한국의 검사기법의 특징을 고찰하였다. 마지막으로 향후 전세계가 '심각한 급성 호흡기 증후군 코로나 바이러스 2'에 대한 동일한 진단결과를 얻기 위하여 코로나-19 진단에 대한 표준화된 진단방법 및 결과판독 등을 제언하였다.