• Journal of the korean academy of Pediatric Dentistry
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• v.35 no.3
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• pp.494-503
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• 2008

Real-time PCR 기법을 이용하여 구강내 치태에서 특정 세균의 존재여부를 알 수 있을 뿐 아니라 정량적 분석이 가능하게 되었다. 이 연구는 real-time PCR 기법을 이용하여 8-18세의 청소년에서 치주질환 원인균의 조성을 알아보고자 전신질환이 없는 어린이 및 청소년 65명을 대상으로 치은연하의 치태를 채취하여 5종의 치주질환 원인균의 출현율과 그 양을 측정하고, 또한 함께 측정한 치태지수 및 치은지수와의 상관관계를 조사하여 다음과 같은 결론을 얻었다. 1. 치태지수의 평균은 1.33이었으며, 치은지수의 평균은 0.97이었다. 치태지수는 나이가 증가할수록 낮아졌다(p<0.05). 2. 치주질환 원인균의 출현율은 P. gingivalis 61.5%, T. forsythia 53.8%, T. denticola 29.2%, A. actinomycetemcomitans 15.4%, F. nucleatum은 100%였다. P. gingivalis, T. denticola는 10세 이후 출현율이 증가하였고 A. actinomycetemcomitans는 8-10세 군에서 출현율이 높았다(p<0.05). 3. 정량적 분석에서 치태지수와 치은지수는 F. nucleatum의 양과 유의한 상관성이 있었으며, 치태지수는 치은지수와 유의한 상관성이 있었다. T. forsythia는 A. actinomycetemcomitans, T. denticola양과 강한 상관성을 보였으며, T. denticola양과 A. actinomycetemcomitans도 강한 상관관계를 가지고 있었다.

• 보존과학연구
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• s.32
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• pp.171-183
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• 2011

In this study molecular genetic analysis was carried out on 4 human skeletal remains from Osuri, Buyeo. We showed that real-time PCR is the method of the choice to assess the initial number of genuine ancient DNA molecules. Human mitochondrial DNA quantification was accomplished by the real-time PCR for the cytochrome b gene of the mitochondria. Histological results proved to be a good potentiality for biochemical analysis using biomolecule. The level of specimen's preservation state was proved that level of quantitative result was BO-04, BO-01, BO-03, BO-02. Continually, we showed that biochemical and biomolecule results for the level of preservation state were similar. This study will be useful to important material for predicting biochemistry and biology analysis of the ancient bone.

• Journal of Korean Society of Water and Wastewater
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• v.26 no.4
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• pp.591-598
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• 2012

Denitrification is an important biological mechanism in wastewater treatment process because this process is technically to remove nitrogen from water to air. There have been lots of study about denitrification engineering and molecular biological research about denitrifying bacteria, respectively. However, combination of these researches was unusual and rare. This study is about the correlation between quantity of denitrifying bacteria and denitrification potential, and consists of NUR batch test as analysis method of denitrification potential and quantitative molecular analysis for denitrifying bacteria. Three reactors (A/O, MLE and A/O of nitrogen deficiency) are operated to get activated sludge with various denitrification potential. All samples which were acquired from reactors were measured denitrification potential by NUR test and NUiR test. Also, Real-time PCR was conducted for quantification of denitrifying bacteria composition in activated sludge. The various denitrification potentials were measured in the reactors. The denitrifiaction potential was the highest in MLE process and the reactor of the nitrogen deficiency showed the lowest. Genomic DNA of activated sludge was obtained and consequently, real-time PCRuse the primer sets of nirK and nirS　were conducted to quantify genes involving denitrification reductase production. As the result of real-time PCR, nirK gene showed more significant influence on denitrification potential comapred with nirS gene.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.132-137
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• 2008

Differential gene expression profiling was carried out in the hepatic tissue of medaka fish, Oryzias latipes, after exposure to an organophosphorus pesticide (OPP), Iprobenfos (IBP), a widely used pesticide in agri- and fish-culture, using a medaka cDNA micro array. Twenty six kinds of differentially expressed candidate genes, with 15 and 11 induced and repressed in their gene expressions, respectively, were associated with cytoskeleton (3.8%), development (7.7%), immune (7.7%), metabolism (30.8%), nucleic acid/protein binding (42.3%) and reproduction (7.7%). Of these genes, changes at the transcription level of five were re-evaluated by real-time quantitative PCR (qRT-PCR). Considering the known function of authentic genes, the effects of IBP on the biological activity and pathological aspects in medaka fish were discussed. The identified genes could be used as molecular biomarkers for biological responses to OPPs contamination in an aquatic environment.

• Journal of Microbiology and Biotechnology
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• v.21 no.10
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• pp.1069-1072
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• 2011

A real-time PCR assay method was established to monitor Leuconostoc spp. populations via specific amplification of the dextransucrase gene. Quantification of L. mesenteroides B-512F using both genomic DNA and cell suspensions yielded a log-linear correlation spanning approximately 5 log units. By using this method, monitoring changes of Leuconostoc spp. during sauerkraut fermentation was successfully accomplished with accuracy after inoculation of starter and sugars (sucrose and maltose).

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1044-1050
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• 2014

Since there is no consensus about the most reliable assays to detect invasive aspergillosis from samples obtained by minimally invasive or noninvasive methods, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis. Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, $200{\pm}20g$) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. The A. fumigatus DNA detection sequence was detected in the following number of samples from 12 immunosuppressed, infected rats examined on the scheduled days: day 1 (0/12), day 3 (0/12), day 5 (6/12), and day 7 (8/12) post-infection. The sensitivity and specificity of the qRT-PCR assay was 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct (cycle threshold) cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal Ct cut-off value was 1.40 (AUC, 0.919). The GM assay was more sensitive than the qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

• Research in Plant Disease
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• v.30 no.3
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• pp.219-228
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• 2024

Pepper anthracnose, caused by Colletotrichum spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of Colletotrichum spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

• Journal of Korean Society of Environmental Engineers
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• v.31 no.10
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• pp.919-926
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• 2009

Enrichment of anaerobic ammonium oxidation (ANAMMOX) bacteria is the essential step for operating full-scale ANAMMOX bioreactor because adding a significant amount of seeding sludge is required to stabilize the ANAMMOX reactor. In this study, the enrichment of ANAMMOX bacteria from an activated sludge using sequencing batch reactor was conducted and verified by analyzing changes in the microbial community structure. ANAMMOX bacteria were successfully enriched for 70 days and the substrate removal efficiencies showed 98.5% and 90.7% for $NH_4\;^+$ and $NO_2\;^-$ in the activity test, respectively. The phylogenetic trees of Planctomycetes phylum showed that the diverse microbial community structure of an activated sludge was remarkably simplified after the enrichment. All 36 clones, obtained after the enrichment, were affiliated with ANAMMOX bacteria of Candidatus Brocadia (36%) and Candidatus Anammoxoglobus (64%) genera. The quantification using real-time quantitative PCR (RTQ-PCR) revea ed that the 16S rDNA concentration of ANAMMOX bacteria was 74.8% compared to the granular ANAMMOX sludge obtained from an upflow ANAMMOX sludge bed reactor which had been operated for more than one year. The results of molecular analysis supported that the enriched sludge could be used as a seeding sludge for a full-scale ANAMMOX bioreactor.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.1
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• pp.11-23
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• 2009

Chromosomal rearrangements are major pathology in hematological malignancies. The detection of minimal residual disease (MRD) for these gene rearrangements helps in monitoring treatment outcomes and predicting prognosis of patients. Recently, quantification of these gene transcripts based on real-time quantitative polymerase chain reaction (RQ-PCR) has been used as MRD detection. The purpose of this study is to ensure the usefulness of the RQ-PCR technique for detecting MRD in hamatological malignancy patients. The patients had been diagnosed to AML1-ETO positive AML, PML-RARa positive AML and BCR-ABL positive MPN at Chonnam National University Hwasun Hospital from Jan. 2006 to Aug. 2008. The fusion transcript was quntified by RQ-PCR and analyzed in comparison to conventional cytogenetics, FISH and RT-PCR. The fusion gene transcript was quantified by RQ-PCR in 57 samples from 14 patients with AML1-ETO positive AML, 79 samples from 27 patients with PML-RARa positive AML and 108 samples from 36 patients with CML. At diagnosis, the quantitative fusion transcripts for AM1-ETO, PML-RARa and BCR-ABL showed the range of 0.485552651~10.82233683 (mean 3.782217131, SD 2.998052348), 0.005300395~0.29267494 (mean 0.056901315, SD 0.080131381) and 0.1293929~12.94826849 (mean 1.701935665, SD 2.200913158). The increase of AML1-ETO fusion gene transcripts preceded morphologic relapse in two patients. Quantification of fusion gene transcripts by RQ-PCR could detected MRD in samples which were negative by in cytogenetic analysis or FISH. Our findings indicated that quantitative analysis of AML1-ETO, PML-RARa and BCR-ABL transcripts by RQ-PCR might be a useful tool for the monitoring of minimal residual disease in hematological malignancies.

• The Korean Journal of Malacology
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• v.27 no.4
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• pp.387-393
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• 2011

The objective of this study was to develop a real-time PCR method for the rapid detection and quantification of the protozoan pathogen Perkinsus olseni using a TaqMan probe. For the standard, genomic DNA was extracted from $10^5$ in vitro-cultured P. olseni trophozoites, and then 10-fold serial dilutions to the level of a single cell were prepared. To test the reliability of the technique, triplicates of genomic DNA were extracted from $5{\times}10^4$ cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an $Exicycler^{TM}$ 96 real-time quantitative thermal block. For quantification, the threshold cycle ($C_T$) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the $C_T$ value and the log concentration of cells in the standard ($r^2$ = 0.996). Detection of DNA at a concentration as low as the equivalent of a single cell showed that the assay was sensitive enough to detect a single cell of P. olseni. The estimated number of P. olseni cells was similar to the original cell concentrations, indicating the reliability of P. olseni quantification by real-time PCR. Accordingly, the designed primers and probe may be used for the rapid detection and quantification of P. olseni from clam tissue, environmental water, and sediment samples.