• BMB Reports
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• 제37권5호
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• pp.546-551
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• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• 산업기술연구
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• 제28권A호
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.184-190
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• 1999

The use of the polymerase chain reaction (PCR) method was described using two sets of primers based on the ceuN gene (JEJ 1 and JEJ 2) which encodes a protein involved in siderophore transport and 16S rRNA gene (pA and pB) for the sensitive and specific detection of enteropathogen Campylobacter jejuni. Six oligonucleotides were utilized in an amplification experiment and PCR products of predicted sizes were generated from whole cells and boiled cell lysates at the same intensity. Two sets of the primer pairs, JEJ and pAB, were specific enough for all C. jejuni strains tested for the direct use of whole cells without DNA extraction or lysis steps. In the PCR using the pAB primer pair, the detection limit, as determined by the ethidium bromide staining of the amplification products on agarose gels, was at the level of $10^1$ bacteria cells or less in both the pure culture and artificially inoculated milk and chicken enrichment samples, whereas the detection limit with the JEJ primer pair was relatively low, i.e. $10^3$ cells or more in the same PCR samples. The PCR method using either a primer JEJ or pAB was both repeatable and specific for the detection of C. jejuni in food. This method is simply completed within 4 h.

• 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.

• KSBB Journal
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• 제29권5호
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• pp.361-371
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• 2014

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.

• 한국동물위생학회지
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• 제25권2호
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• pp.135-140
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• 2002

The purpose of this study was to detect Mycobacterium bovis in cattle(serum, milk, lung, lymph node) by PCR. Nineteen samples from 7 skin test-positive cattle were analyzed. The amplified band of IS6110 by PCR was detected from 2 samples in lung and Iymph node. But the sensitivities of the present methods for detecting M bovis in milk and serum are deficient. Because the PCR sensitivity has been shown to be hindered by the method used to isolate the nucleic acid target. PCR-based methods have the potential to be faster, more accurate, and the most efficient means of detecting M bovis. The detection of M bovis by PCR will contribute to the more efficient detection and control of tuberculosis.

• The Plant Pathology Journal
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• 제31권1호
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• pp.25-32
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• 2015

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

• The Plant Pathology Journal
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• 제22권2호
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• pp.164-167
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• 2006

An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.

• 미생물학회지
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• 제41권3호
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• pp.168-176
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• 2005

갯벌 퇴적물에 존재하는 병원성 해양미생물인 Vibrio vulnificus를 신속하고 정확하게 검출하기 위해 PCR, Southern hybridization 방법과 real-time PCR을 수행하여 검출 민감도를 비교하였다. 갯벌 퇴적물로부터 bead beater를 이용한 물리적 방법으로 DNA 조추출액을 얻고 상용화된 키트 (Geneclean turbo Kit)를 이용하여 부식물질(humic substances)을 제거하였다. 병원성에 관련된 3 종의 유전자(hemolysin, vvhA; phosphomannomutase, pmm; metalloprotease, vvpE)를 대상으로 설계한 프라이머 셋을 동시에 사용하는 multiplex PCR 방법과 Southern hybridization과 병행한 방법(PCR/Southern hybridization)을 수행하였다. Real-time PCR은 hemolysin 유전자(vvhA)에 특이한 프라이머와 TaqMan 탐침을 사용하였다. 전처리하지 않은 갯벌 퇴적물의 경우, PCR/Sourthern hybridization과 real-time PCR 방법의 검출 민감도는 퇴적물 1 g 당 약 $10^2$ 개의 세포 수준이었다. 농후처리액(APW; alkaline peptone water)으로 $35^{\circ}C$에서 $2{\~}3$시간, 8시간 중균 배양할 경우 갯벌 퇴적물 1 g 당 $2{\~}10$개 세포가 존재할 때 PCR/Southern hybridization 방법과 real-time PCR 방법으로 각각 검출할 수 있었다. 전처리 과정을 포함하여 real-time PCR은 $6{\~}7$시간, PCR/Sourthern hybridization은 약 36시간이 소요되었다.

• 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.