• 한국임상수의학회지
• /
• 제14권2호
• /
• pp.177-184
• /
• 1997

Canine parvovirus(CPV) is a very highly contagious virus causing hemorrhagic enteritis and myocarditis mainly in young dogs. The diseases were first recognized in 1978, and then spread throughout the world by 1980. The main source of the infection seems to be the feces of infected dogs, at the same time feces are suitable materials for detection of virus in the enteric form exactly for the same reasons. Recently, a new technique of in vitro DNA amplification, Known as the polymerase chain reaction (PCR), has been widely applied to clinical viral diagnosis because of its sensitivity, specificity and rapidity. In this research, we attemped to set up the PCR for the detection of CPV in fecal samples and conformed the canine parvpviral enteritis by PCR. To increase the sensitivity and specificity of a PCR, the nested PCR (two-step PCR) was performed. We also surveyed the contamination status of CPV in the research using fecal specimen was highly sensitive and specific. Of the 100 fecal specimens suspected canine parvoviral enteritis, 45 fecal specimens were positive in HA test, 64 fecal specimens were positive in the first PCR, and 87 fecal specimens were positive in the second PCR. CPV contamination status of animal clinics and breeding centers was serious, wo hygienic management of environment in which dogs are reared is required. The nested PCR described here seems to be a rapid, sensitive and specific for the detection of canine parvovirus.

• 한국동물위생학회지
• /
• 제40권3호
• /
• pp.161-168
• /
• 2017

Caprine arthritis encephalitis (CAE) virus is a causative agent of caprine arthritis-encephalitis. In our previous study we reported a prevalence of CAE. In this study, we described the further detailed pathological features of CAE and examined the detection of virus by in situ hybridization (ISH). Histopathologically, interstitial pneumonia and bronchopneumonia in lung, focal inflammation in mammary glands, perivascular cuffing in brain, arthritis, and focal necrosis, mild steatosis, inflammatory cell infiltration of liver were noted. CAEV proviral-DNA was identified by nested polymerase chain reaction (PCR) in blood cells, brain, synovial fluid, and lymph node. Confirmation by nested PCR involved amplification of a 296 bp ($1^{st}$ PCR) and 185 bp ($2^{nd}$ PCR) fragments corresponding to a conserved region on the gag gene of CAEV. Positive ISH signals were detected in the brain and liver. In conclusion, significant histopathological findings included parenchymal infection in various organs, including the lung, liver, brain, joint, and mammary gland were noted in the CAEV infected dairy goat. ISH can help confirm the diagnosis of CAE in formalin-fixed samples.

• 미생물학회지
• /
• 제43권3호
• /
• pp.179-185
• /
• 2007

본 연구는 polymerase chanin reaction(PCR)기법과 DNA enzyme-linked immunosorbent assay(DNA ELISA) 기법을 이용하여 토양내 식물병원균인 Ralstonia solanacearum를 검출하고자 하였다. 토양 시료로부터 분석에 사용될 R. solanacearum DNA를 추출하기 위하여 몇 가지 방법을 비교 평가한 결과 기존의 DNA 추출 방법에 비하여 Guanidin isothiocyanate와 Chelex-100 resin을 사용하는 방법 이 토양 내에 존재하는 다양한 중류의 반응 저해 물질과 R. solanacearum만의 고유한 PCR반응 저해물질들을 제거하는 데에 효과적이었다. R. solanacearum만을 특이적으로 검출하기 위해 fliC유전자 부위에 특이적인 몇 종의 primer들을 제작하였다. 이들 중 높은 민감도와 특이도를 나타내는 두 set의 primer RsolfliC(forward; 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.)와 RS_247 (forward; 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3, designed by this study)를 선정하여 nested PCR을 수행할 수 있도록 고안하였다. Nested PCR primer에 biotin을 표지하였고 nested PCR산물의 내부 서열과 특이적으로 교잡반응을 할 수 있는 probe를 제작하여 PCR 결과를 DNA-EIA반응으로 확인 분석할 수 있도록 하였다. Primary PCR과 nested PCR의 산물을 전기영동 상에서 확인한 결과, nested PCR이 약 $10^2$정도의 높은 민감도를 나타내었고 DNA-EIA의 경우 $10^2P{\sim}10^3$정도의 민감도를 상승시켜주는 것으로 확인되었다.

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

• 생태와환경
• /
• 제54권3호
• /
• pp.190-198
• /
• 2021

사람 아이치바이러스(Aichivirus A; AiV-A)는 positivesense single-strand RNA 비외피 바이러스로 지난 10년 동안 하수, 강, 지표 및 지상의 다양한 물환경에서 전 세계적으로 검출이 보고되고 있다. 지하수 등 물환경에서 AiV-A 진단을 위한 고감도 및 특이성이 우수한 방법의 개발이 요구됨에 따라, 본 연구에서는 기존 및 신규 설계된 프라이머 세트를 기초로 역전사(RT) 및 이중 중합효소연쇄반응이 가능한 조합을 개발하였다. 개발한 방법을 국내 음용 지하수 시료에 적용 및 평가하였으며, 그 결과 지하수 시료에서 AiV-A를 성공적으로 검출 및 동정할 수 있는 RT-nested PCR primer sets가 선정되었고 후속적으로 동정할 수 있는 절차가 고안되었다. 본 연구 결과는 지하수 등 물 환경에서 AiV-A 오염을 탐지하기 위한 모니터링 시스템 마련에 기여할 것으로 기대된다.

• 대한의생명과학회지
• /
• 제24권4호
• /
• pp.390-397
• /
• 2018

For the specific detection of human Parvovirus B19 (HuPaV-B19), we designed ten specific PCR primers from 3,800~4,500 nucleotides of HuPaV-B19 complete genome (NC_000883.2). Seventeen candidate PCR primer sets for specific detecting HuPaV-B19 were constructed. In specific reaction of HuPaV-B19, seventeen PCR primer sets showed specific band, however five PCR primer sets were selected basis of band intensity, amplicon size and location. In non-specific reaction with seven reference viruses, four PCR primer sets showed non-specific band, however one PCR primer set is not. Detection sensitivity of final selective PCR primer set was $100fg/{\mu}L$ for 112 minute, and PCR amplicon is 539 base pairs (bp). In addition, nested PCR primer set was developed, for detection HuPaV-B19 from a low concentration of contaminated samples. Selection of nested PCR primer set was basis of sensitivity and groundwater sample tests. Detection sensitivity of final selective PCR and nested PCR primer sets for the detection of HuPaV-B19 were $100fg/{\mu}L$ and $100ag/{\mu}L$ basis of HuPaV-B19 plasmid, it was able to rapid and highly sensitive detection of HuPaV-B19 than previous reports. We expect developed PCR primer set in this study will used for detection of HuPaV-B19 in various samples.

• Food Science and Biotechnology
• /
• 제18권2호
• /
• pp.407-412
• /
• 2009

In this study, a rapid and efficient concentrating procedure that can be used for detecting viruses in vegetables was developed. The Sabin strain of poliovirus type 1 was used to evaluate the efficiency of virus recovery. The procedure included: (a) elution with 0.25 M threonine-0.3 M NaCl pH 9.5; (b) polyethylene glycol (PEG) 8000 precipitation; (c) chloroform extraction; (d) 2$^{nd}$ PEG precipitation; (f) RNA extraction; (g) reverse transcription-polymerase chain reaction (RT-PCR) combined with semi-nested PCR. The overall recoveries by elution/concentration were 29.0% from cabbage and 13.7% from lettuce. The whole procedure usually takes 18 hr. The overall detection sensitivity was 100 RT-PCR units of genogroup II norovirus (GII NoV)/25 g cabbage and 100 RT-PCR units of GII NoV/10 g lettuce. The virus detecting method developed in this study should facilitate the detection of low levels of NoV in cabbage and lettuce.

• 한국식품과학회지
• /
• 제31권6호
• /
• pp.1628-1634
• /
• 1999

Listeria monocytogenes의 식품 속에서 신속하고 특이적인 검출을 위하여, listeriolysin O gene에 의한 primer, LM 1과 LM 2를 선택하여 PCR을 수행하였다. L. monocytogenes의 DNA 추출이나 cell lysis 없이 intact whole cell을 직접 이용하여 PCR을 하였으며 $10^{2-6}$ CFU 수준의 균체 배양액으로부터 L. monocytogenes에 특이적인 702 bp의 PCR 증폭 산물을 확인하였다. 우유, 닭고기, 김치 등의 식품에 L. monocytogenes를 접종하여 증균배양 전후 균의 PCR에 대한 감도와 생균수를 비교한 결과, 실험된 식품 속에서의 L. monocytogenes의 검출 감도는 순수 배양액에서의 경우에 비해 약 1/10로 둔화되었으나 역시 특이적인 검출이 가능하였다. Primer LM 1과 2를 이용한 본 실험조건에서의 L. monocytogenes의 PCR에 의한 검출은 약 4시간으로 확인이 가능하였으며, 기존의 배양 방법에 비해, 특이성이나 검출 속도 면에서 식품위생 실무에 적용하기 위한 높은 잠재력을 보여 주었다.

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

• 대한의생명과학회지
• /
• 제27권4호
• /
• pp.255-263
• /
• 2021

Human Astrovirus (HuAstV) is an important gastrointestinal pathogen that is frequently reported worldwide. Monitoring of contaminated groundwater has been suggested since HuAstV is transmitted through the fecal-oral route. This study developed a test method based on conventional reverse transcription (RT)-nested polymerase chain reaction (PCR) that involves SL^® non-specific reaction inhibitor for unknown non-specific amplification taking place in the groundwater environment. An optimal method for detecting HuAstV in groundwater sample through analysis and comparison against conventionally reported method was also suggested. The developed method enabled the production of nested PCR amplicon of 630 nt, which is a sufficient length for similarity analysis based on sequencing and genotyping. Amplicons suspected to be HuAstV were amplified in two out of the twenty groundwater samples collected in Korea, presenting 99.77% and 99.73% similarity against HuAstV 1 strain lhar/2011/kor (JN887820.1) in sequencing, respectively. These amplicons were identified as HuAstV 1.