• Journal of Microbiology
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• 제38권4호
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• pp.260-264
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• 2000

Reverse transcription (RT)-PCR and nested PCR methods (2-step PCR) were tested for their ability to detect marine birnavirus (MBV) in cultured rockfish, Sebastes schlegeli. One set of primers for RT-PCR was designed, based on a gene of infectious pancreatic necrosis virus (IPNV), and another set of primers for nested PCR was designed based on the VP2/NS junction region of MBV. This 2-step PCR method was specific for MBV and sensitivity was heightened when nested PCR was combined to RT-PCR. This 2-step PCR method was useful for detecting MBV not only in diseased fish, but also in asymptomatic fish. These results indicate that this 2-step PCR method is useful for detecting MBV in rockfish.

• 대한바이러스학회지
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• 제27권1호
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• pp.49-57
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• 1997

We developed a sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) to detect Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in animal tissues. Total RNA was extracted from blood, lung or kidney samples of experimentally-infected hamsters by using the guanidine isothiocyanate buffer-acid phenol-chloroform method. Genus-reactive outer primers were derived from the consensus region of the G1 gene sequences of several hantaviruses. Serotype-specific primers were selected within the region amplified by the outer primers. To examine the sensitivity and specificity of the test, we diluted known quantities of Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses in human or hamster immune sera before performing the nested RT-PCR. We could detect as little as 1 pfu of virus, even in the presence of high-titer neutralizing antibodies, and the serotype-specific primers amplified only homologous serotype viruses. RT-PCR with these primers demonstrated virus in the blood of experimentally-infected hamsters as early as four days to as late as 30 days after infection. A comparison of a standard immunofluorescent antibody screening test (IFAT) to nested RT-PCR with RNA extracted from lung or kidney tissues of the hamsters, demonstrated that RT-PCR to be more sensitive for identifying viruses in these tissues.

• Parasites, Hosts and Diseases
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• 제48권2호
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• pp.99-103
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• 2010

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.

• Journal of Genetic Medicine
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• 제9권1호
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• pp.11-16
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• 2012

Purpose: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. Materials and Methods: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. Results: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. Conclusion: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

• 식물병연구
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• 제16권2호
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• pp.125-128
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• 2010

자두 주산지인 경북 김천을 비롯하여 의성, 경산, 군위지역에 발생하는 자두과실의 검은점무늬병 병원균을 조기 검출하기 위하여 integrons를 이용한 primer를 제작하고 DNA 추출향상을 위해 전배양법을 그리고 검출감도 향상을 위해 nested PCR을 시도하였다. 제작된 integrons primer로 병반 시료를 PCR한 결과 예상되는 크기인 760 bp로 증폭이 되어 검은점무늬병의 원인 병원균은 Xanthomonas arboricola pv. pruni로 확인되었고 진단용 primer로 사용 될 수 있음을 확인할 수 있었다. DNA 추출 방법별로 PCR 진단효율은 조사한 결과 직접 추출하는 방법보다는 10% LB배지로 전배양한 다음 DNA를 추출하는 방법이 더 효과적이었고 nested PCR을 이용할 경우에는 과원내 강우중의 X. arboricola pv. pruni의 검출도 가능하였다. 이번에 개발된 Integrons primer를 이용, 전배양법과 nested PCR을 실시할 경우 1일 이내에 병징이 나타나지 않은 유묘에서의 X. arboricola pv. pruni 감염여부나 식물검역에서의 X. arboricola pv. pruni 감염식물 진단 등에도 활용이 가능할 것으로 생각된다.

• 한국임상수의학회지
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• 제17권1호
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• pp.13-20
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• 2000

Recently cases of canine distemper have occurred in Korea despite vaccination was carried out nationwidely. This study was performed to establish rapid diagnosis of canine distemper by RT-PCR, nested PCR, and serological test. A total of 30 dogs, which were suspected canine distemper clinically, was examined. RT-PCR and nested PCR were specific for the amplification of CDV H gene and sensitive to detect 7 TCID50 of Onderstepoort strain. By RT-PCR, H gene was detected in 6(20%) of 30 peripheral bloods from dogs. And H gene was detected in 10(33.3%) of 30 samples by nested PCR. H gene was detected from 1 brain of 6 years-old Beagle dog and 1 lung of 2 months-old Shihtzu dog, in which peripheral blood H gene was not detected. Serum neutralizing antibody titer against Onderstepoort strain ranged from 4 to 1,024 in 30 patients. No correlation was observed between the results of nested PCR and titiers of neutralizing antibody.

• 대한바이러스학회지
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• 제30권3호
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• pp.179-187
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• 2000

The etiology of rheumatic arthritis (RA) is associated with a number of genetic and environmental factors, but is not definitively elucidated. Recently, more attention has been paid to the possibility of microbial etiology in the pathogenesis of RA, because many different infectious agents have been reported to precede the onset or exacerbation of RA. Adenovirus (ADV) may be one cause of persistent or recurrent inflammatory arthritis. Varicella zoster virus (VZV) arthritis is detected frequently in RA patients treated with low dose methotrexate. The demonstration of simultaneous presence of both viral agents of specific viral nucleic acid in synovial fluids from synovitis patients would provide more direct evidence for arthritis etiological relationship, but there are no confirmed results. Therefore, we studied the ability of adenovirus and VZV to establish coinfection and persistent infection in synovial fluid from synovitis patients. The presence of viral agents in the synovial fluid demonstrated by isolation of cell culture, enzyme immunoassay and nested-PCR. The synovial fluids were also investgated for the presence of viral nucleic acid by nested-PCR using specific primer. ADV produced 220 bp and VZV produced 447 bp by each nested-PCR with specific primers. We detected 4/6 cases (66.7%) with persistent infection of ADV and 5/6 cases (83.3%) of VZV with 13 synovial fluids (between 7 to 52 day intervals) from synovitis patients by monoclonal ErA and nested-PCR. 21/28 cases (75%) with coinfection of adenovirus and VZV with synovial fluids from synovitis patients by nested-PCR. ADV and VZV coinfection and persistent infection of synovial fluids may provide a chronic antigenic stimuli to the immune system therefore provoking a continuing inflammatory response and caused the possibility of synovitis and arthritis.

• 식물병연구
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• 제9권3호
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• pp.116-120
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• 2003

참다래 잎으로부터 궤양병균인 Pseudomnoas syringae pv. actinidiae를 배양하여 PCR을 통해 검출하는 방법을 개발하였다. Nested PCR을 위해 두 set의 primer를 식물 독소인 coronatine의 생합성에 관여하는 유전자 cfl의 염기서열로부터 설계하였다. 이 primer set를 사용하여 665rhk 310-bp의 절편이 증폭되었으며 nested PCR을 통한 궤양병균의 검출한계는 20 CFU/ml이었다. 4 그루의 참다래 나무로부터 노란색 무리로 나타나는 궤양병 초기 증상을 보이는 잎을 채취하여 pepton-sucrose 액체배지에 넣어 $16^{\circ}$C에서 12시간 배양 한뒤 PCR 을 시행한 결과 한 시료에서 예상했던 밴드가 증폭되었고 이듬해 봄 이 나무가 궤양병에 감염되었음을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.3003-3007
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• 2015

Objective: To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Materials and Methods: Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). Results: The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p<0.05). Patients with early EOC had markedly lower serum CA125 than those with advanced EOC (p<0.05), but there was no significant difference in free DNA methylation (p>0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (p<0.05). In the detection of patients with advanced EOC, the PPV of CA125 detection was obviously lower than that of multiplex nested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Conclusions: Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.

• 대한수의학회지
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• 제42권2호
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• pp.225-229
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• 2002

We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.