• 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.

• BMB Reports
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• 제33권1호
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• pp.59-62
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• 2000

Hepatitis C virus (HCV) nonstructural 5B (NS5B) protein is an RNA-dependent RNA polymerase (RdRp). To determine whether it can contribute to viral replication by interaction with cellular proteins, the yeast two-hybrid screening system was employed to screen a human liver cDNA library. Using the HCV NS5B as a bait, we have isolated positive clones encoding a cellular protein. The NS5B interacting protein, 5BIP, is a novel cellular protein of 170 amino acids. Interaction of the HCV NS5B protein with 5BIP was confirmed by a protein-protein blotting assay. Recently, we have demonstrated that NS5B possesses an RdRp activity and thus it is possible that 5BIP, in association with NS5B, plays a role in HCV replication.

• Journal of Microbiology and Biotechnology
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• 제5권4호
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• pp.181-187
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• 1995

The cells of Vibrio vulnificus can be induced to the viable but nonculturable (VBNC) state by natural environmental parameters. The V. vulnificus cells in the VBNC state can not be recovered by ordinary laboratory techniques. This nonculturability could often hamper development of effective processing strategies to minimize the number of V. vulnificus in seafoods. Even with V. vulnificus cells in a culturable state, the length of time required to identify the bacteria in contaminated food by phenotyphic characterization may prevent appropriate in-time responses by public health agencies to infections of the bacteria. In the present study, we used polymerase chain reaction (PCR) to develop a rapid and direct detection method for V. vulnificus in small octopus (Octopus variabilis) which is consumed as a raw food in Korea. The region targeted was a 704-base pair (bp) portion of the hemolysin gene, vvhA, of V. vulnificus. The primers designed for PCR amplification were specific for all V. vulnificus sp. tested. Several methods were examined to extract total DNA directly from V. vulnificus seeded into the octopus homogenate and the guanidine isothiocyanate (CITC) method appeared to be most effective. From the octopus homogenate seeded by V. vulnificus at an initial level of $10^2$ CFU/ml of the homogenate and then incubated for 12 h, the targeted sequence was successfully amplified by PCR and the 704-bp DNA fragment was observed by gel electrophoresis. The total completion of this assay requires less than one day.

• Asian Pacific Journal of Cancer Prevention
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• 제14권9호
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• pp.4989-4992
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• 2013

Aim: To compare p16INK4a immunocytochemistry with the HPV polymerase chain reaction in predicting high grade cervical squamous intraepithelial lesions. Materials and Methods: This diagnostic case-control study was conducted from January 2010 until December 2010. We obtained 30 samples, classified according to the degree of cervical intraepithelial neoplasia (CIN): 11 samples for CIN 1, 9 samples for CIN 2, and 10 samples for CIN 3. HPV PCR, p16INK4a immunocytochemistry, and histopathological examination were performed on all samples. Statistical analysis was conducted using SPSS 20.0. Results: In predicting CIN 2-3, we found p16INK4a to have similar specificity and positive predictive value as HPV PCR (95%, 97.2% vs 96.7%), but better sensitivity (87.5% vs 72.5%) and negative predictive value (82.1% vs 67.6%). The most prevalent types of high-risk HPV in our study were HPV 33, 35, 58, 52, and 16. Conclusions: p16INK4a has better diagnostic values than HPV PCR and may be incorporated in the triage of ASCUS and LSIL to replace HPV PCR. Genotype distribution of HPV differs in each region, providing a challenge to develop HPV vaccines based on the epidemiology of HPV in that particular region.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.801-806
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• 2002

A multiplex competitive polymerase chain reaction (PCR) method was developed for the rapid identification and quantification of Leuconostoc mesnteroides and Lactobacillus plantarum populations which are the key microorganisms in kimchi fermentation. The strain-specific primers were designed to selectively amplify the target genes encoding 165 rRNA of L. plantarum and dextransucrase of L. mesenteroides. There was a linear relationship between the band intensity of PCR products and the number of colony forming units of each model organism. The PCR quantification method was compared with a traditional plate-counting method f3r the enumeration of the two lactic acid bacteria in a mixed suspension culture and also applied to a real food system, namely, watery kimchi. The population dynamics of the two model organisms in the mixed culture were reliably predictable by the competitive PCR analysis.

• Fisheries and Aquatic Sciences
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• 제11권3호
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• pp.133-139
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• 2008

Molecular techniques, including restriction fragment length polymorphism(RFLP) and polymerase chain reaction-single strand conformational polymorph isms(PCR-SSCP), were developed to identify salted, dried threadfin(Eleutheronema tetradactylum) and white herring(Ilisha elongata) fish. Using PCR with universal primers, conserved 367-bp fragments of the cytochrome b gene were amplified from fresh fish samples and sequenced. The sequences were then searched for specific restriction sites. The digestion of the PCR products with the endonucleases AvaI, FokI, MboII, and MspI generated RFLP, which was used to identify the commercial products. Similarly, the amplified PCR-SSCP products were developed and the products tested. Overall, similar patterns were found in the majority of the fresh and processed products. Based on the results, both RFLP and PCR-SSCP were useful in determining and validating the authenticity of the fish species used to prepare the commercial salted, dried products. A similar approach can be applied to other species.

• Molecules and Cells
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• 제21권3호
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• pp.330-336
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• 2006

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, ${\alpha}$-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.

• 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.

• Pediatric Infection and Vaccine
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• 제3권2호
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• pp.207-213
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• 1996

Herpes simplex virus(HSV) infections of the CNS are associated with significant morbidity and mortality even when appropriate antiviral therapy is administered. HSV infections of the brain can be subdivided into two categories : neonatal HSV infections, which usually are caused by HSV type 2, and herpes simplex encephalitis(HSE), which occur in patients over 3 months old and is nearly uniformly caused by HSV type 1. The clinical presentation of HSE is one of the focal encephalopathic process associated with altered levels of consciousness, fever, focal seizures and hemiparesis. But because of the lack of pathognomic clinical presentation and diagnostic procedure, the efforts to develop alternative diagnostic procedure have led to the use of new diagnostic technique such as polymerase chain reaction(PCR). We report a case of HSV type 1 encephalitis in 13 month old male infant who presented with altered level of consciousness, fever and focal seizures. With the use of the PCR, HSV-1 DNA was detected in cerebrospinal fluid from the patient. The symptoms and signs of encephalitis subsided by treatment with acyclovir in 14 days.