• Journal of Microbiology
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• v.34 no.3
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• pp.229-235
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• 1996

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

• Journal of Microbiology
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• v.44 no.2
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• pp.162-170
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• 2006

Oysters are known to be carriers of food-born diseases, but research on viruses in Korean oysters is scarce despite its importance for public health. We therefore tested oysters cultivated in Goheung, Seosan, Chungmu, and Tongyeong, for viral contamination using cell culture and integrated cell culture PCR (ICC-PCR) with Buffalo green monkey kidney (BGMK) and human lung epithelial (A549) cells. Additional screens via PCR, amplifying viral nucleic acids extracted from oysters supplemented our analysis. Our methods found 23.6 %, 50.9 %, and 89.1 % of all oysters to be positive for adenoviruses when cell culture, ICC-PCR, and direct PCR, respectively, was used to conduct the screen. The same methodology identified enteroviruses in 5.45%, 30.9%, and 10.9% of all cases. Most of the detected enteroviruses (81.3%) were similar to poliovirus type 1; the remainder resembled coxsackievirus type A1. A homology search with the adenoviral sequences revealed similarities to adenovirus subgenera C (type 2, 5, and 6), D (type 44), and F (enteric type 40 and 41). Adenovirus-positive samples were more abundant in A549 cells (47.3%) than in BGMK cells (18.2 %), while the reverse was true for enteroviruses (21.8 % vs. 14.5 %). Our data demonstrate that Korean oysters are heavily contaminated with enteric viruses, which is readily detectable via ICC-PCR using a combination of A549 and BGMK cells.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.1
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• pp.17-23
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• 2015

A fine needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant in thyroid nodules. However, between 10 and 30% of the FNABs of thyroid nodules are diagnosed as 'indeterminate'. A molecular method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is classic papillary type and BRAFV600E mutation is highly prevalent. Thus, this study compared the pyrosequencing method with the conventional direct DNA sequencing and PCR-RFLP analysis and investigated the evaluation of preoperative BRAFV600E mutation analysis as an adjunct diagnostic method with routine FNABs. Sixty-five (78.3%) of 83 histopathologically diagnosed malignant nodule revealed positive BRAFV600E mutation on pyrosequencing analysis. In detail, 65 (83.8%) of 78 papillary thyroid carcinomas sample showed positive BRAFV600E mutation. None of 29 benign nodules had in pyrodequencing, direct DNA sequencing and PCR-RFLP. Out of 31 thyroid nodules classified as 'indeterminate' on cytological examination preoperatively, 28 cases turned out to be malignant: 24 papillary thyroid carcinomas. Among that, 16 (66.7%) classic papillary thyroid carcinomas had BRAFV600E mutation. Among 65 papillary thyroid carcinomas with positive BRAFV600E mutation detected by pyrosequencing analysis, each 3 cases and 5 cases did not show BRAFV600E mutation by direct DNA sequencing and PCR-RFLP analysis. Therefore, pyrosequencing was superior to direct DNA sequencing and PCR-RFLP in detecting the BRAFV600E mutation of thyroid nodules (p =0.027). Detecting BRAFV600E mutation by pyrosequencing was more sensitivity, faster than direct DNA sequencing or PCR-RFLP.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1481-1483
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• 2010

We constructed an efficient T-vector, pTQEST216T that employed an engineered esterase as an indicator for direct cloning of PCR products. After ligation of the XcmI-digested vector with PCR products, this cloning system could easily discriminate positive clones owing to insertional inactivation of the esterase reporter. Additionally, PCR products were efficiently cloned into this vector without the gel purification steps, owing to the well-designed multi-cloning site that was in-frame fused at the circularly permutated gap of the reporter.

• Mycobiology
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• v.38 no.1
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• pp.33-38
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• 2010

We have successfully applied the nested PCR to detect Cylindrocarpon destructans, a major pathogen causing root rot disease from ginseng seedlings in our former study. The PCR assay, in this study, was used to detect the pathogen from soils. The nested PCR using internal transcribed spacer (ITS) 1, 4 primer set and Dest 1, 4 primer set maintained the specificity in soils containing various microorganisms. For a soil DNA extraction method targeting chlamydospores, when several cell wall disrupting methods were tested, the combination of lyophilization and grinding with glass beads, which broke almost all the chlamydospores, was the strongest. The DNA extraction method which was completed based on the above was simple and time-saving because of exclusion of unnecessary stages, and efficient to apply in soils. As three ginseng fields whose histories were known were analyzed, the PCR assay resulted as our expectation derived from the field information. The direct PCR method will be utilized as a reliable and rapid tool for detecting and monitoring C. destructans in ginseng fields.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1470-1474
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• 2009

This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.1
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• pp.49-55
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• 2007

HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5'UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5'UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes.

• Research in Plant Disease
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• v.15 no.2
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• pp.83-87
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• 2009

Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X axonopodis pv. glycines with DNA concentration as low as 200 fg and $1.8{\times}10^3$ cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.196-202
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• 2006

Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However the most troublesome problem of lamivudine treatment is the emergence of lamivudine-resistant strains with amino acid substitution in the YMDD motif of DNA polymerase gene during the treatment. The aim of this study was to determine the mutation of YMDD motif (codon 552) and codon 528 in chronic HBV patients with lamivudine therapy using PCR-direct sequencing and to investigate the relationship between lamivudine mediated HBV mutation and HBeAg. HBV DNA was extracted from serum samples of HBV patients and amplified by nested PCR with two sets of primer pairs selected in HBV DNA polymerase gene. Amplified PCR product was analyzed by 2% agarose gel electrophoresis and direct sequencing. HBV mutation was detected in 124 out of 207 samples (60%). Single mutation was 50.8% for M552I, 43.5% for M552V, 5.7% for M552I/V and the L528M mutation was 67.0%. Double mutation was 43.6% for M552V/L528M, 33.1% for M552I/L528(wild type), 17.7% for M552I/L528M and 5.6% for M552I/V/L528M. Serine mutation at YMDD motif (M552S) was not found and the L528M mutation frequently accompanied M552V type. In this study, the typical difference of frequencies for HBV mutation depending on HBeAg was not found. Moreover, the PCR-direct sequencing method used in this study might be a powerful tool for the mutation study in clinical reference laboratories with high volume.

• BMB Reports
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• v.30 no.3
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• pp.234-236
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• 1997

Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain $10^{5}-10^{6}$ copies of HCV RNA in 1 ml of serum.