• 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.

• Journal of Microbiology and Biotechnology
• /
• 제18권8호
• /
• pp.1453-1458
• /
• 2008

For quantitative PCR assay of Salmonella enterica serovar Typhimurium in food samples, a real-time PCR method was developed, based on DNA genome equivalent. Specific primers and probe designed based on the STM4497 gene of S. Typhimurium LT2 showed the specificity to S. Typhimurium. Threshold cycle (Ct) values of real-time PCR were obtained from a quantitative standard curve with genomic DNA of Salmonella Typhimurium. In addition, the recovery of S. Typhimurium inoculated artificially to chicken samples with $4.5{\times}10^5$ to 4.5 CFU/ml was evaluated by using real-time PCR and plate-count methods. Result showed that the number of cells calculated from the real-time PCR method had good correlation with that of the plate-count method. This real-time PCR method could be applicable to the detection and quantification of S. Typhimurium in food samples.

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

• 한국환경농학회지
• /
• 제30권4호
• /
• pp.367-376
• /
• 2011

논 토양의 미생물 생태 다양성을 조사하기 위한 효과적인 방법으로 qRT-PCR을 적용하고자 본 연구를 수행하였다. 논 토양 미생물의 gDNA를 분리하기 위하여 Mo Bio kit를 사용한 효과적이고 안정적인 gDNA 분리 방법을 확립하였다. 논 토양 미생물 다양성을 qRT-PCR로 검출하기 위하여 bacteria를 세분한 ${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes 다섯 가지 문과 전체 bacteria, 전체 fungi를 구분할 수 있는 특이 primer set을 선정하여 다양한 조건의 시험을 통하여 최종 조건을 확립하였으며 재현성 실험을 통하여 방법의 유의성을 검증하였다.

• International Journal of Oral Biology
• /
• 제46권3호
• /
• pp.140-145
• /
• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• Journal of Microbiology and Biotechnology
• /
• 제15권6호
• /
• pp.1353-1358
• /
• 2005

A rapid and quantitative real-time PCR was developed to target the invasion A (invA) gene of Salmonella spp. We developed quantitative standard curves based on plasmids containing the invA gene. Based on these curves, we detected Salmonella spp. in artificially contaminated buffered peptone water (BPW) and milk samples. We were able to determine the invA gene copy number per ml of food samples, with the minimum detection limit of $4.1{\times}10^{3}$ copies/ml of BPW and $3.3{\times}10^{3}$ copies/ml of milk. When applied directly to detect and quantify Salmonella spp. in BPW and milk, the present real-time PCR assay was as sensitive as the plate count method; however, copy numbers were one to two logs higher than the colony-forming units obtained by the plate count methods. In the present work, the real-time PCR assay was shown to significantly reduce the total time necessary for the detection of Salmonella spp. in foods and to provide an important model for other foodborne pathogens.

• 한국미생물·생명공학회지
• /
• 제33권2호
• /
• pp.148-153
• /
• 2005

말라리아는 세계적으로 다시 발생하는 감염성 질환으로 모기에 의해 옮겨지는 말라리아 원충에 의해서 발병한다. 말라리아 원충을 검출하기 위하여 혈액 도말법 등 여러 가지 정량적인 assay가 활용 되고 있다. Real time PCR 은 반응이 일어나는 동안 PCR 산물의 생성을 계속적으로 모니터링 할 수 있는 방법으로서 최근 많은 환자들의 말라리아 원충을 한번에 탐지 하는데 적용되고 있다. 본 연구에서는 말라리아 원충 중 한국에서 질환을 일으키는 Plasmodium vivax를 탐지하기 위해 26명의 환자 혈액에서 분리 정제한 genomic DNA를 가지고 SYBR Green based-real time PCR을 수행하였다. 특히, 기존의 real time PCR에 사용한 18S rRNA와는 달리 DBP 유전자를 사용하여 새로운 말라리아 검출방법을 정량적이면서도 간편하고 경제적인 방법으로 개발하였다. 특히, real time PCR로 나온 결과를 임상적인 titer로 바꾸어 주기 위해서 reference 유전자로는 말라리아 환자의 상태와 관계없이 ACTB이 안정하다는 것을 real time PCR로 입증하였고 ACTB 유전자를 reference 유전자로 사용하였다. 본 연구에서는 real time PCR assay에 DBP라는 유전자를 처음 사용하였을 뿐 아니라 titer 보정을 위한 reference 유전자, ACTB를 real time PCR assay을 통해 확보하여 더 정확한 titer 값을 얻고자 했다. 이런 결과를 바탕으로 Taqman based-real time PCR과 본 연구의 결과를 정량비교를 하였다. 특히, 26명의 말라리아 환자 샘플을 3 group(Group I, II, III)로 나누어서 그 결과 정량적인 경향성 일치를 나타내었다. 특히, 높은 titer을 갖는 말라리아 샘플(Group I)에서 가장 많은 정량적인 차이를 보였지만, 간편하고 경제적인 SYBR Green-based real time PCR을 이용하여 DBP 유전자를 증폭하는 새로운 방법으로 말라리아를 Semi-quantitative 하게 검출할 수 있음을 보였다.

• 한국환경보건학회지
• /
• 제45권5호
• /
• pp.511-519
• /
• 2019

Objectives: The standard method for the enumeration of environmental Legionella is culturing, which has several disadvantages, including long incubation and poor sensitivity. The purpose of this study is to demonstrate the usefulness of real-time PCR and to improve the standard method. Methods: In 200 environmental water samples, a real-time PCR and culture were conducted to detect and quantify Legionella. Using with the results of the survey, we compared the real-time PCR with the culture. Results: Each real-time PCR assay had 100% specificity and excellent sensitivity (5 GU/reaction). In the culture, 36 samples were positive and 164 samples were negative. Based on the results of the culture, real-time PCR showed a high negative predictive value of 99%, 35 samples were true positive, 105 samples were true negative, 59 samples were false positive and one sample was a false negative. Quantitative analysis of the two methods indicated a weak linear correlation ($r^2=0.29$, $r^2=0.61$, respectively). Conclusions: Although it is difficult to directly apply quantitative analysis results of real-time PCR in the enumeration of environmental Legionella, it can be used as a complementary means of culturing to rapidly screen negative samples and to improve the accuracy of diagnosis.

• Journal of Microbiology and Biotechnology
• /
• 제18권4호
• /
• pp.648-657
• /
• 2008

In this study, we used functional genomic strategies, proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production in the fungus Fusarium verticillioides. Earlier studies have demonstrated that deletion of the FCC1 gene, which encodes a C-type cyclin, leads to a drastic reduction in fumonisin production and conidiation in the mutant strain (FT536). The premise of our research was that comparative analysis of F. verticillioides wild-type and FT536 proteomes will reveal putative proteins, and ultimately corresponding genes, that are important for fumonisin biosynthesis. We isolated proteins that were significantly upregulated in either the wild type or FT536 via two-dimensional polyacrylamide gel electrophoresis, and subsequently obtained sequences by mass spectrometry. Homologs of identified proteins, e.g., carboxypeptidase, laccase, and nitrogen metabolite repression protein, are known to have functions involved in fungal secondary metabolism and development. We also identified gene sequences corresponding to the selected proteins and investigated their transcriptional profiles via quantitative real-time (qRT)-PCR in order to identify genes that show concomitant expression patterns during fumonisin biosynthesis. These genes can be selected as targets for functional analysis to further verify their roles in $FB_1$ biosynthesis.

• 한국어병학회지
• /
• 제31권2호
• /
• pp.101-107
• /
• 2018

여윔증상이 나타난 양식장(farm-B 및 farm-C)의 넙치 및 여윔증상이 나타나지 않은 양식장(farm-A) 넙치의 각 내부 장기(신장, 장, 비장, 뇌 및 간)를 대상으로 점액포자충 Parvicapsula sp.의 양적 분석을 real-time PCR 방법을 사용하여 각각 실시하였다. 여윔증상을 보였던 farm-C의 넙치 신장에서 가장 높은 DNA copy number ($1.7{\times}10^7copies/mg$ tissue)를 보였고, farm-B의 넙치에서는 모든 내부 장기에서 낮은 수치가 나타났으며, farm-A의 넙치에서는 모든 내부 장기에서 음성 결과를 나타내었다. 동일한 시료를 사용한 PCR 및 병리조직학적 분석에서도 real-time PCR에서의 결과와 같은 양상을 보였다.