• Environmental Engineering Research
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• 제20권1호
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• pp.51-57
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• 2015

As expected, the expression of denitrifying genes in a Typha wetland (relatively stagnant compared to other ponds), showing higher nitrogen removal efficiency in summer, was affected by temperature. The abundance and gene transcripts of nitrate reductase (narG), nitrite reductase (nirS), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ) genes in seasonal sediment samples taken from the Acorus and Typha ponds of free surface flow constructed wetlands were investigated using quantitative polymerase chain reaction (Q-PCR) and quantitative reverse transcription PCR (Q-RT-PCR). Denitrifying gene copy numbers ($10^5-10^8$ genes $g^{-1}$ sediment) were found to be higher than transcript numbers-($10^3-10^7$ transcripts $g^{-1}$ sediment) of the Acorus and Typha ponds, in both seasons. Transcript numbers of the four functional genes were significantly higher for Typha sediments, in the warm than in the cold season, potentially indicating greater bacterial activity, during the relatively warm season than the cold season. In contrast, copy numbers and expression of denitrifying genes of Acorus did not provide a strong correlation between the different seasons.

• 대한수의학회지
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• 제38권3호
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• pp.559-564
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• 1998

The polymerase chain reaction(PCR) assay was used for rapid diagnosis from blood and organ samples experimentally infected with Listeria monocytogenes. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L monocytogenes. Procedure A was based on dilution of the blood sample followed by lysis of bacterial cell and direct analysis of the lysate with PCR. In artificially infected blood samples with L monocytogenes, it was possible to detect fewer than 40 cells per ml of blood. However, L monocytogenes was detected low rates on infected organs by the direct PCR. In procedure B, enrichment cultivation was used to increase numbers of bacteria before lysis and PCR. L monocytogenes was detected from 23 samples of 24 liver and spleen, respectively, and 18 samples of 24 blood were found to be positive by PCR on a subset of 72 organ samples, whereas L monocytogenes were detected on 63 organ samples in classical culture technique. It was required to analyze including enrichment steps were 6h and 18h on the procedure A and B, respectively.

• 한국식품위생안전성학회지
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• 제23권4호
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• pp.314-318
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• 2008

This research aimed to compare the detection methods of Anisakis simplex in Sea fish by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and macroscopic inspection. We examined 18 Trichiurus lepturus, 11 Scomber japonicus, and 65 Todarodes pacificus collected from the retail markets in the areas of Uljin, Kyuonggi province and Seoul. As the result of examinations, we found that detection rate of Anisakis simplex by macroscopic observation was 89% in Trichiurus lepturus, 90.9% in Scomber japonicus, 32.3% in Todarodes pacificus. The detection rate of Anisakis simplex by PCR-RFLP was 77.7% in Trichiurus lepturus, 81.8% in Scomber japonicus, 26.1% in Todarodes pacificus. We could conclude that PCR-RFLP method of Anisakis simplex was more specific rather than macroscopic observation.

• 한국축산식품학회지
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• 제37권4호
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• pp.599-605
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• 2017

Korean native honey (KNH) is much more expensive than European honey (EH) in Korea, because KNH is a favored honey which is produced less than EH. Food fraud of KNH has drawn attention of the government office concerned, which is in need of a method to differentiate between KNH and EH which are produced by the Asiatic honeybee, Apis cerana and the European honeybee, Apis mellifera, respectively. A method to discriminate KNH and EH was established by using duplex polymerase chain reaction (PCR) in this study. Immunochromatographic assay (IC) was examined to analyze the duplex PCR product. The DNA sequences of primers for the duplex PCR were determined by comparing cytochrome C oxidase genes of the two honey bee species. Chelex resin method was more efficient in extracting genomic DNA from honey than the other two procedures of commercial kits. The duplex PCR amplifying DNA of 133 bp were more sensitive than that amplifying DNA of 206 bp in detecting EH in the honey mixture of KNH and EH. Agarose gel electrophoresis and IC detected the DNA of 133 bp at the ratios of down to 1% and 5% EH in the honey mixture, respectively and also revealed that several KNH products distributed by internet shopping sites were actually EH. In conclusion, the duplex PCR with subsequent IC could also discriminate between KNH and EH and save time and labor.

• 한국미생물·생명공학회지
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• 제22권6호
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• pp.577-583
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• 1994

To classify Lactobacillus casei strains on the basis of difference in their chromosomal DNA sequence, we have performed polymerase chain reactions on their chromosomal DNA by using random primers, and followed by analyzing randomly amplified polymorphic DNA fragments. We also developed a mini-preparative method to isolate PCR-grade chromosomal DNA from Lacto- bacillus casei strains within 3 hours. Based on RAPD pattems by polymerase chain reactions with degenerated random primers, 4 Lactobacillus casei strains and 2 mutant strains were successfully discriminated. Results were very sensitive, strain-specific and reproducible. It was also reliable. These results suggest that RAPD may be applied efficiently for the identification of several Lactoba- cillus casei strains.

• Journal of Plant Biotechnology
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• 제5권2호
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• 한국동물위생학회지
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• 제39권2호
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• pp.107-116
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• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

• 한국수산과학회지
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• 제47권6호
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• pp.740-744
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• 2014

In this study, the ${\beta}$-lactamase (VPA0477) gene was used as a new target for the PCR-based detection of Vibrio parahaemolyticus. Primers specific for the ${\beta}$-lactamase (VPA0477) gene of V. parahaemolyticus, were designed and incorporated into a PCR-based assay. The assay was able to specifically detect all of the 191 V. parahaemolyticus strains tested, but did not result in amplification of 39 other Vibrio spp. and non-Vibrio spp. strains tested. The detection limit of the assay was 10 CFU of V. parahaemolyticus RIMD2210633 from pure culture broth. The ${\beta}$-lactamase (VPA0477) gene-based assay developed in this study was sensitive and specific, and has great potential for the accurate detection and identification of V. parahaemolyticus in seawater or seafood samples.

• 대한임상검사과학회지
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• 제40권2호
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• pp.94-97
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• 2008

The human leukocyte antigen (HLA) is the name of the major histocompatibility complex (MCH) in humans. The superlocus contains a large number of genes related to immune system function in humans. This group of genes resides on chromosome 6. and encode cell surface antigen-presenting proteins and many other genes. HLA class I antigen (A, B & C) present peptides from inside the cell. These peptides are produced from digested proteins that are broken down in the lysozymes. Most expressed HLA loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino terminal domain of the molecule. In this sutdy, the HLA-A genotypes were determined in twenty students unrelated koreans using the PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primer) technique. Several specific primer pairs in assigning the HLA-A gene were used (A*0201, A*33, A*2401). The results of PCR-SSP, the HLA-A*0201 primer was detected eleven (55%), the HLA-A*33 were detected seven (35%) and the HLA-A*2401 were detected seven (35%). This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-A genotypes.

• 한국잠사곤충학회지
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• 제37권2호
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• pp.167-171
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• 1995

딱정벌레목 천공성 해충인 뽕나무 하늘소(Apriona germari)의 효과적인 방제를 위하여, 뽕나무 하늘소 이병충으로 부터 곤충병원 사상균을 분리하고, 위상차 현미경 및 주사전자현미경으로 관찰하였으며, PCR(polymerase chain reaction)을 이용하여 동정하였다. 뽕나무 하늘소 이병충으로 부터 분리된 곤충병원 사상균은 현미경 관찰 결과 Beauveria속의 전형적인 형태적 특성을 나타냈다. 따라서 이들의 용이한 동정을 위하여 PCR primer(5'-ACG GGC GCT C-3')를 이용한 RAPD(random amplification of polymorphic DNA) 방법으로 분석하고, B. bassiana와 B. brongniartii의 PCR 산물을 전기영동한 결과 DNA 표식자로 이용이 가능하였다. 이상의 결과로서 본 실험에서 분리·명명된 SFB-1A는 B. bassianifh, SFB-1A는 B. bassiana로, SFB-3A는 B. brongniartii로 동정되었다.