• Journal of Microbiology
• /
• 제41권4호
• /
• pp.320-326
• /
• 2003

In order to identify Vibrio vulnificus in the Yellow Sea near Gunsan, Korea during the early and late summers, the efficiency of the real-time quantitative TaqMan PCR was compared to the efficiency of the conventional PCR and Biolog identification system^TM. Primers and a probe were designed from the hemolysin/cytolysin gene sequence of V. vulnificus strains. The number of positive detections by real-time quantitative TaqMan PCR, conventional PCR, and the Biolog identification system from seawater were 53 (36.8%), 36 (25%), and 10 strains (6.9%), respectively, among 144 samples collected from Yellow Sea near Gunsan, Korea. Thus, the detection method of the real-time quantitative TaqMan PCR assay was more effective in terms of accuracy than that of the conventional PCR and Biolog system. Therefore, our results showed that the real-time TaqMan probe and the primer set developed in this study can be applied successfully as a rapid screening tool for the detection of V. vulnificus.

• Food Science and Biotechnology
• /
• 제17권3호
• /
• pp.651-654
• /
• 2008

In order to enhance the efficacy of norovirus detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, this study developed a norovirus mRNA concentration method using poly oligo dT-conjugated magnetic beads. An efficient norovirus detection protocol was performed on commercial ham using 2 viral elution buffers (glycine buffer and Tris beef extract buffer) and 2 concentration solutions [polyethylene glycol (PEG) and zirconium hydroxide]. The different approaches were verified by RT-PCR and nested PCR. This method was performed on ham in less than 8 hr by artificial inoculation of serial dilutions of the virus ranging from 1,000 to 1 RT-PCR unit/mL. The viral extraction and concentration method had 10-fold higher sensitivity using the combination of Tris beef extract buffer and PEG as compared to glycine buffer and zirconium hydroxide. This method proved that RT-PCR and nested PCR have the sensitive ability to detect norovirus in commercial ham, in that norovirus was successfully detected in artificially contaminated samples at a detection level as low as 1-10 RT-PCR unit/mL. Overall, such a detection limit suggests this protocol is both quick and efficient in terms of its potential use for detecting norovirus in meat products.

• 대한수의학회지
• /
• 제44권1호
• /
• pp.99-103
• /
• 2004

The aim of the present study was to develop a sensitive and specific assay for the diagnosis of alcelaphine herpesvirus 1 (AlHV-1) which is a cause agent of malignant catarrhal fever in ruminants. A1HV-1 is a gamma herpesvirus, which is frequent latent, and it is often difficult to detect its antigens or specific nucleic acids because of its low genomic copies in the infected tissues. In this study, polymerase chain reaction (PCR)-dot blot hybridization (DBH) assay for detecting AlHV-1 DNA was developed and evaluated for its sensitivity and specificity as comparison with PCR and DBH alone. The developed PCR-DBH was more sensitive than PCR or DBH alone and also very specific. The results showed that the sensitivity of PCR-DBH were higher and stronger than those of PCR and DBH alone. This PCR-DBH assay can be applied efficiently to confirm the presence of AlHV-1 virus on clinical samples and to differentiate specifically between AlHV-1 infection and other viral infections.

• Journal of Microbiology and Biotechnology
• /
• 제19권11호
• /
• pp.1464-1469
• /
• 2009

To achieve more accurate and rapid detection of macrolide-lincosamide-streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

• 미생물학회지
• /
• 제38권2호
• /
• pp.103-106
• /
• 2002

사상균류의 세포자체로부터 직접 PCR을 하는 방법은 세포벽 파괴의 어려움 때문에 모든 곰팡이에 적용될 수 없다. 극초단파 조사는 사상균류의 DNA를 추출함에 있어서 그 유용함이 이미 검중된 바 있는데, 본 논문에서는 극초단파 조사를 이용하여 Aspergillus nidulans의 포자로부터 손쉽게 주형 DNA를 얻어 PCR증폭하는 방법을 소개하고자 한다. 본 실험에서는 극초단파 조사시간과 PCR에 필요한 주형 DNA의 양 등을 최적화하였으며, 이렇게 수행된 PCR결과는 single copy유전자를 대상으로도 약 3 kb크기의 산물가지 증폭 가능하여, 형질전환체를 선별하기에 충분한 크기의 산물들도 효과적으로 얻어짐을 보여주었다. 따라서 이 방법은A. nidulans의 형질전환체를 보다 손쉽게, 시간을 절약하여 선별할 수 있는 효과적인 방법이라 생각된다.

• 대한의생명과학회지
• /
• 제24권3호
• /
• pp.230-238
• /
• 2018

Human enteric Adenovirus-41 (HuEAdV-41) causes gastroenteritis, which detected by the polymerase chain reaction (PCR) base diagnostic system for clinical, food, environmental, fish and shellfish samples. We developed improved PCR and nested PCR primer set which had high specificity, sensitivity and reduced times. In this study, we compared seventeen conditions reported in the previous study that was using the PCR based HuEAdV-41 detection system, and non-enteric Adenovirus were detected in nine conditions. The most sensitive detection condition was up to 25 copies however it took 184 minutes of PCR reaction time. In this study, the PCR primer set developed had same level of sensitivity, it reduced the time of detection for clinical, food and seafood samples to 112 minutes. Developed nested PCR primer set needed 112 minutes but detected up to approximately 1 copy. In addition, developed PCR and nested PCR primer set was validated with twenty samples of underground water at random, of which ten samples showed specific band without non-specific reaction. We expect this study will be used to diagnose HuEAdV-41 from various samples.

• 한국식품과학회지
• /
• 제33권5호
• /
• pp.521-524
• /
• 2001

본 연구는 유전자재조합 기술에 의해 개발된 glyphosate에 내성을 가지고 있는 콩(GTS)의 모니터링을 위하여 PCR을 이용한 검출 방법에 대한 실험을 수행하였다. Glyphosate에 내성이 있는 콩에 삽입된 유전자와 표준대조 유전자인 lectin과 ferritin 유전자를 근거로 제작된 primer와 CTAB 방법으로 추출된 콩의 DNA를 template로 이용하여 PCR을 수행하였다. GTS의 검출을 위한 제작된 primer들은 GTS와 특이적으로 반응하여 증폭된 PCR 산물을 생성하였으나, non-GTS와는 PCR 산물을 생성하지 못했다. 증폭된 염기서열 분석을 통하여 GTS에 특이적인 것을확인하였으며, 약 0.05%가 포함되어 있는 GTS까지 검출이 가능함을 보였다.

• 한국미생물·생명공학회지
• /
• 제45권4호
• /
• pp.358-364
• /
• 2017

Asymmetric PCR preferentially amplifies one DNA strand for use in DNA hybridization studies. Linear-After-The-Exponential-PCR (LATE-PCR) is an advanced asymmetric PCR method which uses innovatively designed primers at different concentrations. This study aimed to optimise LATE-PCR parameters to produce single-stranded DNA of Candida spp. and Aspergillus spp. for detection via probe hybridisation. The internal transcribed spacer (ITS) region was used to design limiting primer and excess primer for LATE-PCR. Primer annealing and melting temperature, difference of melting temperature between limiting and excess primer and concentration of primers were optimized. In order to confirm the presence of single-stranded DNA, the LATE-PCR product was hybridised with digoxigenin labeled complementary oligonucleotide probe specific for each fungal genus and detected using anti-digoxigenin antibody by dot blotting. Important parameters that determine the production of single-stranded DNA in a LATE-PCR reaction are difference of melting temperature between the limiting and excess primer of at least $5^{\circ}C$ and primer concentration ratio of excess primer to limiting primer at 20:1. LATE-PCR products of Candida albicans, Candida parapsilosis, Candida tropicalis and Aspergillus terreus at up to 1:100 dilution and after 1 h hybridization time, successfully hybridised to respective oligonucleotide probes with no cross reactivity observed between each fungal genus probe and non-target products. For Aspergillus fumigatus, LATE-PCR products were detected at 1:10 dilution and after overnight hybridisation. These results indicate high detection sensitivity for single-stranded DNA produced by LATE-PCR. In conclusion, this advancement of PCR may be utilised to detect fungal pathogens which can aid the diagnosis of invasive fungal disease.

• 식물병연구
• /
• 제16권2호
• /
• pp.125-128
• /
• 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.

• 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.