• 대한바이러스학회지
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• 제27권2호
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• pp.169-176
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• 1997

In this study, the feasibility of identification and genotypic differentiation of enteroviruses was investigated by using nested reverse transcription-polymerase chain reaction (nested RT-PCR), single-stranded conformation polymorphism (SSCP), and restriction fragment length polymorphism (RFLP) techniques. Two hundred seventy-four clinical samples were assayed by both nested RT-PCR and tube culture method using MRC-5 and MK cells; 58 (86.6%) out of 67 enterovirus culture-positive samples contained enteroviral RNA. In addition, 114 (55.1%) of 207 samples from patients with suspected enteroviral CNS disease with negative viral cultures were positive by the nested RT-PCR. The nested RT-PCR products were genotyped by the SSCP method and the results were compared with serotypes. We could differentiate 6 subtypes, 3 of which are similar to coxsackievirus B3, B5, echovirus 11, plus 3 other subtypes. RFLP cleaved with Sty I, Bgl I, and Xmn I yielded characteristic patterns for each laboratory strains. This study demonstrates the usefulness of the RT-PCR for the rapid diagnosis of enterovirus infection and the potentials of the SSCP method for differentiation of enterovirus strains.

• Parasites, Hosts and Diseases
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• 제52권4호
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• pp.377-381
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• 2014

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.

• Parasites, Hosts and Diseases
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• 제49권3호
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• pp.281-284
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• 2011

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

• 대한바이러스학회지
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• 제26권2호
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• pp.235-244
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• 1996

Rickettsia tsutsugamushi의 원형균주인 Karp주와 Gilliam주를 초대 배양된 사람 정상 2배체 폐세포(LuMA cell)를 이용하여 증식과 세포병변들의 속도를 비교할 수 있었고, 배양된 균주는 네스티드 프라이머를 사용하여 혈청형을 동정할 수 있었다. R. tsutsugamushi의 세포벽 외막에 존재하며 혈청형을 결정하는 주요항원은 54-56Kd 단백인 것으로 밝혀지고 있는데, 이 단백 유전자의 DNA 염기서열을 분석하여 Karp주와 Gilliam주의 공통서열로 첫번째 프라이머쌍을 만들었고 첫번째 프라이머쌍의 안쪽에 위치한 혈청형 사이에 차이가 있는 서열로 두번째 프라이머쌍을 만들었다. 네스티드 뉴클레오티드 프라이머는 중합효소 연쇄반응의 특이성을 증가시킬 수 있는데 이 실험 결과로 이 PCR 방법은 scrub typhus의 진단과 혈청형의 동정에 적용될 수 있을 것으로 보여진다.

• Tuberculosis and Respiratory Diseases
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• 제72권2호
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• pp.156-162
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• 2012

Background: The main goal of this study was to evaluate the diagnostic efficacy of reverse transcription-nested polymerase chain reaction (RT-nested PCR) in bronchial washing fluid with MAGE A1-6 common primers for the detection of lung cancers invisible by bronchoscopy. Methods: To determine the expression of MAGE A1-6 gene in 189 lung cancers diagnosed by conventional fluoroscopy-guided lung biopsy and 89 cancer-free controls, RT-nested PCR was performed in bronchial washing specimens. We analyzed MAGE A1-6 RT-nested PCR data according to tumor histology, stage, size, and compared them with cytological data. Results: 189 patients (111 cases in adenocarcinoma, 47 cases in squamous cell carcinoma, 22 cases in small cell lung carcinoma, and 9 cases in other cancers) and 89 benign patients were investigated. The expression of MAGE was performed by nested RT-PCR using common MAGE primer. Among 189 cancer patients, the expression rate of MAGE was 49.2%, and the positive predictive value was 89.4%. However, the expression rate of MAGE in patients with benign lesions was 12.4%. In peripheral lung cancer, the positive rate of MAGE expression was 57.4% in squamous cell carcinoma, 44.1% in adenocarcinoma and 59.1% in small cell lung cancer. Whereas the expression rate of bronchial washing cytology in peripheral lung cancer was 9.0% (p=0.011). Conclusion: MAGE RT-PCR in bronchial washing fluid gave us promising data for the detection of peripheral lung cancer. It could be a useful method for selecting diagnostic tools for peripheral lesions.

• Journal of Microbiology
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• 제44권1호
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• The Plant Pathology Journal
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• 제30권2호
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• pp.178-182
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• 2014

Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea.

• 대한의생명과학회지
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• 제25권4호
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• pp.331-338
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• 2019

Human Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

• 한국식품과학회지
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• 제44권5호
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• pp.638-642
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• 2012

돼지축사에서 채집해온 6개의 육성돈의 분변에서 식중독 유발 바이러스인 HAV와 HEV를 검출하였으며, HAV는 88.3%의 검출율을 보였으며, HEV는 33.3%의 검출율을 보였다. 결과에는 제시하지 않았으나, 염기서열 분석결과 HEV는 사람에게 전염이 가능한 유전자형인 III형이었으며, 실험적으로 사람의 간세포인 PLC/PRF/5에 접종하였을 때 증식이 됨을 확인하였다. 식중독 유발 바이러스인 HAV와 HEV는 오염된 식품이나 물을 섭취하거나 교차 오염에 의해 전염이 가능하기 때문에 돼지축사에서 위생상태의 개선뿐만 아니라 육류를 섭취하기 전인 운송 및 가공과정까지 식중독 유발 바이러스에 의한 교차오염을 막는 노력이 필요하다. HAV와 HEV 모두 검출된 분변에서 HAV를 순수분리하고 빠르게 검출하기 위해 IMS-RT-PCR을 적용하였으며, 항원-항체 반응에 의해 순수하게 HAV만을 분리할 수 있었다. 또한 HAV만이 순수분리 되었는지 재확인하기위해 세포감염을 통해 증식된 바이러스를 확보한 후 nested RT-PCR을 수행한 결과, HAV만을 순수 분리할 수 있음을 확인하였다. 이는 IMS 활용기술이 단순히 항체를 교체함으로써 다른 특정 식중독 유발 바이러스의 다양한 시료에서 바이러스 순수분리 및 검출에 활용 가능성이 있음을 확인하였다.

• 한국수정란이식학회지
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• 제13권2호
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• pp.97-106
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• 1998

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% $CO_2$ incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.