• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1164-1169
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• 2008

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus-negative specimens. Furthermore, the assays could detect and subtype up to $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.816-824
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• 2017

Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

• The Plant Pathology Journal
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• 제18권1호
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• pp.12-17
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• 2002

For the molecular detection of Sweet potaio feathery mottle virus (SPFMV) from diseased sweet potato plants, reverse transcription and polymerase chain reaction (RT-PCR) was performed with the use of a set of virus-specific primers to amplify an 816 bp product. The viral coat protein gene was selected for the design of the primers. No PCR product was amplified when Turnip mosaic virus, Potato vims Y or Cucumber mosaic virus were used as template in RT-PCR with the SPFMV-specific primers. The lowest concentration of template viral RNA required for detection was 10 fg. The vim was rapidly detected from total nucleic acids of leaves and roots from the virus-infected sweet potato plants as well as from the purified viral RNA by the RT-PCR. Twenty-four sweet potato samples were selected and analyzed by RT-PCR and restriction fragment length polymorphism (RFLP). RFLP analysis of the PCR products showed three restriction patterns, which resulted in some point mutations suggesting the existence of quasi-species for the vims in the infected sweet potato plants.

• 한국미생물·생명공학회지
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• 제36권1호
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• pp.34-42
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• 2008

소의 혈액, 세포, 조직, 기관 등 소 유래 물질을 원료로 사용한 생물의약품, 조직공학제제, 세포치료제의 경우 소 유래원료 물질에 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. BVDV는 동물세포주, 우혈청 등에 가장 흔하게 오염되는 바이러스이다. 소 유래물질을 원료로 하는 생물의약품, 조직공학제제, 세포치료제 등에서 BVDV안전성을 확보하기 위해, 원료물질, 제조공정, 완제품에서 BVDV를 정량적으로 검출하고, 제조공정에서 BVDV제거 검증을 위한 시험법으로 활용이 가능한 BVDV real-time RT-PCR시험법을 확립하였다. BVDV에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 BVDV RNA 정량 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time RT-PCR 민감도는 $1TCID_{50}/mL$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성 (specificity)과 재현성(reproducibility)이 우수함을 확인하였다. 확립된 real-time RT-PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 BVDV를 오염시킨 CHO 세포주와 소 유래콜라겐에서 BVDV검출 시험을 실시하였다. BVDV를 감염시킨 CHO 세포와 세포배양 상청액에서 BVDV를 정량적으로 검출할 수 있었다. 소 유래 콜라겐에서도 $10TCID_{50}/mL$까지 정량적으로 검출할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.685-689
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• 2000

Due to the uncultivable nature of Mycobacterium leprae in vitro, the fast, easy, and accurate measurement of the antimicrobial drug susceptibility of this microbe has been difficult. Conventional methods for such testing are subjective, cumbersome, and expensive in some cases. Here, the utility of a reverse transcriptase-PCR (RT-PCR)-based assay for testing was examined and compared with a Buddmeyer-type radiorespirometric assay. The susceptibility of M. leprae to rifampin was determined by probing the presence of M.leprae-specific 18 kDa gene mRNA in M. leprae-infected IC-21 macrophage cells after drug treatment. The results showed that, as the refampin concentration was increased, the 360-bp cDNA products generated by the RT-PCR-based assay decreased in a dose-dependent manner as in the drug susceptibility observed in the Buddmeyer-type assay. The drug susceptibility testing of M. leprae by the RT-PCR based assay was found to be not only faster but also nearly $10^4$-fold more sensitive than the Buddmeyer-type assay. Moreover, it was also found that, unlike the RT-PCR based assay, the same testing by a DNA-PCR resulted in no differences in the 360-bp signal, regardless of the rifampin concentrations used. Accordingly, these results demonstrated that the drug susceptibility of M. leprae can be determined effectively by an RT-PCR-based assay, thereby providing a new, fast, and sensitive testing method.

• 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.

• 식물병연구
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• 제19권3호
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• pp.220-225
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• 2013

본 연구에서는 식물검역바이러스 4종(TBRV, ArMV, CLRV 및 GFLV)을 RT-PCR과 nested PCR 방법으로 진단 할 수 있는 방법을 개발하였다. 본 연구에서 개발한 방법은 모두 같은 PCR 조건으로 검사자에게 편리성과 신속성을 높여줄 뿐 아니라, 돌연변이-양성대조구의 사용으로 실험 오염여부를 확인할 수 있어 더욱 정확하다. 개발한 방법으로 최근 3년 Nepovirus속 4종의 바이러스를 검사한 결과, 27건을 검출하여 검역처분 하였다. 본 연구 결과들은 앞으로도 수출입 식물에서 해당 바이러스들을 신속, 정밀하게 진단할 수 있는 방법으로 활용할 수 있을 것으로 기대된다.

• 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.

• 대한바이러스학회지
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• 제30권2호
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• pp.151-159
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• 2000

Hepatitis C virus (HCV) is one of the important human pathogen that can cause acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Recently, the third generation radiation immuno assay (RIA) method has been developed as a very sensitive test to detect anti-HCV antibody. However, false positive is the problem with RIA test. To solve this the RIA results were compared to those of 5-antigen recombinant immunoblot assay (5-RIBA) and reverse transcription-polymerase chain reaction (RT-PCR). Among 12,767 serum samples tested from clinic visitors, total 275 (2.2%) samples were antibody positive by RIA. RIBA was performed with 148 RIA positives cases but among them was shown eighty five was antibody positive and sixty three (42.6%) was negative result. However, nested RT-PCR test was shown also carried out with 43 positive, 6 intermediates and 25 negatives of RIBA. As a result of the nested RT-PCR results, HCV antigen were detected in RIBA positive, 33.3% (2/6) RIBA intermediate and 12% (3/25). Clinical syndrome of all 148 patients as a with chronic active hepatitis (46.0%), cirrhosis (18.9%), hepatocellular carcinoma (8.1%) and others (27.0%) and they were positive in reaction by RIA test. But RIBA positive patients with 34.9% of chronic active hepatitis, 18.6% of cirrhosis, 4.6% of hepatocellular carcinoma and 41.9% of others were detected to be positive case by nested RT-PCR.

• The Plant Pathology Journal
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• 제20권2호
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• pp.142-146
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• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.