• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2733-2737
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• 2014

Objective: Molecular pathology tests are often carried for clinicopathological diagnosis and pathologists have established large collections of formalin-fixed, paraffin-embedded tissue (FFPE) banks. However, extraction of DNA from FFPE is a laborious and challenging for researchers in clinical laboratories. The aim of this study was to compare two widely used DNA extraction methods: using a QIAamp DNA FFPE kit from Qiagen and a Cobas Sample Preparation Kit from Roche, and evaluated the effect of the DNA quality on molecular diagnostics. Methods: DNA from FFPE non-small cell lung carcinoma tissues including biopsy and surgical specimens was extracted with both QIAamp DNA FFPE and Cobas Sample Preparation Kits and EGFR mutations of non-small cell lung carcinomas were detected by real-time quantitative PCR using the extracted DNA. Results and Conclusion: Our results showed that DNA extracted by QIAamp and Cobas methods were both suitable to detect downstream EGFR mutation in surgical specimens. Howover, Cobas method could yield more DNA from biopsy specimens, and gain much better EGFR mutation results.

• Biomedical Science Letters
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• v.15 no.3
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• pp.229-232
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• 2009

DNA isolation for PCR-based short tandem repeat (STR) analysis is essential to recover high yields of amplifiable DNA from low copy number (LCN) DNA samples. There are different methods developed for DNA extraction from the small bloodstain and gloves, commonly found at crime scenes. In order to obtain STR profiles from LCN DNA samples, DNA extraction protocols, namely the automated $iPrep^{TM}$ $ChargeSwitch^{(R)}$ method, the automated $QIAcube^{TM}$ method, the automated $Maxwell^{(R)}$ 16 DNA $IQ^{TM}$ Resin method, and the manual $QIAamp^{(R)}$ DNA Micro Kit method, were evaluated. Extracted DNA was quantified by the $Quantifiler^{TM}$ Human DNA Quantification Kit and DNA profiled by $AmpFISTR^{(R)}$ $Identifiler^{(R)}$ Kit. Results were compared based on the amount of DNA obtained and the completeness of the STR profiles produced. The automated $iPrep^{TM}$ $ChargeSwitch^{(R)}$ and $QIAcube^{TM}$ methoas produced reproducible DNA of sufficient quantity and quality trom the dried blood spot. This two automated methods showed a quantity and quality comparable to those of the forensic manual standard protocols normally used in our laboratory. In our hands, the automated DNA extraction method is another obvious choice when the forensic case sample available is bloodstain. The findings of this study indicate that the manual simple modified $QIAamp^{(R)}$ DNA Micro Kit method is best method to recover high yields of amplifiable DNA from the numerous potential sources of LCN DNA samples.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.775-781
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• 2015

Background: RB1 (retinoblastoma 1) was reportedly one of the major determinative factors for sensitivity to taxanes in previous studies. In this study, we investigated the influence of RB1 single nucleotide polymorphisms (SNPs) on the efficacy of platinum-taxane regimens in advanced NSCLC patients. Materials and Methods: 234 cases of patients with advanced NSCLC who were treated with first-line platinum-taxane agents were enrolled in this study. Genomic DNA was extracted from patients' peripheral blood samples using a QIAamp DNA Maxi Kit, and genotyped by iSelect HD Bead-Chip. Results: Regression analyses were conducted through the univariate and multivariate Cox proportional hazards model in the 234 patients. The results showed that of the eight RB1 tagSNPs, only rs4151510 was a positive predictive factor for the advanced NSCLC patients treated with platinum taxanes regimen. The patients with G/G genotype of RB rs4151510 had longer overall survival (OS) than the non-G/G genotype (p=0.018). The histology was also correlated with OS in the whole advanced NSCLC patients. Three tagSNPs of RB1, rs4151510, rs4151465, rs9568036 were significantly associated with OS in the advanced NSCLC patients with squamous cell histology using Kaplan-Meier overall survival analysis stratified by histology. Conclusions: RB1 genomic variants were correlated with the efficacy of platinum-taxanes regimen. RB rs4151510 is an independent factor of the prognosis of NSCLC patients receiving platinum-taxane chemotherapy.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.263-271
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• 2014

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. $QIAamp^{(R)}$ DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume ($50-100{\mu}l$) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ${\approx}2$ oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.9
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• pp.1097-1105
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• 2017

This study examined how much chicken meat was in sausage made with pork. Both real-time polymerase chain reaction (PCR) and internal standard addition were used. Fifty ng of chicken DNA was added to the sausages as an internal standard. The addition of standard DNA increased the amplification efficiency of PCR and confirmed the possibility of quantitative analysis. A QIAamp DNA Micro Kit was used to improve the DNA recovery and amplification efficiency. The density of template DNA and primer were suitable for $3.0{\sim}5.0{\mu}L$ and $0.5{\mu}L$, respectively. Each DNA of pig and chicken was diluted in 10-fold from steps 50 ng to 0.05 ng. The detection limit of both pig and chicken meat was more than 0.05 ng and the correlation coefficient of the standard curve was at least 0.98. The result of the quantitative analysis after heat treatment of 3 samples of pigs and chickens mixed at 70:30 showed a 5.7% difference (64.3:35.7) between the expected value and measured value. The quantitative value was changed by affecting the DNA according to the heat treatment ($70^{\circ}C$, 10 min). An analysis of the pork and chicken content in sausages showed that it was difficult to detect chicken meat and the quantitative value of DNA according to the Ct value was very low. On the other hand, when adding standard material (50 ng of chicken DNA) to the sausages, the Ct value decreased gradually with increasing chicken mixing ratio. Thus, the mixing ratio of chicken in sausages could be estimated.

• Journal of Food Hygiene and Safety
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• v.30 no.2
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• pp.150-158
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• 2015

This article reports the development of an effective test procedure for detection of norovirus (NoV) in foods of diverse matrices. In this study, target foods included fermented milk, soybean paste, powders made from uncooked grains and vegetables, sesame leaves preserved in soy sauce, pickled mooli, and mooli. Viral recovery varied depending on the food matrices or elution buffers tested. Buffers were compared to determine effective elution buffers from artificially virus-contaminated foods. The conventional test procedure for concentrating viruses from food (elution-polyethylene glycol(PEG) precipitation-chloroform-PEG precipitation) was modified to save time by eliminating one PEG precipitation step. The modified procedure (elution-chloroform-PEG precipitation) was able to concentrate viruses more effectively than the conventional procedure. It also removed RT-PCR inhibitors effectively. The modified procedure was applied to target food for genogroup II NoV detection. NoV RNA was detected at the initial inoculum levels 3.125-12.5 RT-PCR units per 10-25 g tested food. The use of this newly established procedure should facilitate detection of low levels of norovirus in diverse foods.

• Journal of Korean Medicine for Obesity Research
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• v.19 no.2
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• pp.97-105
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• 2019

Objectives: We investigated differences between the tracheostomized and the non-tracheostomized stroke patients through microbiological analysis for the purpose of preliminary explorations of full-scale clinical research in the future. Methods: We collected tracheal aspirates samples from 5 stroke patients with tracheostomy and expectorated sputum samples from 5 stroke patients without tracheostomy. Genomic DNA from sputum samples was isolated using QIAamp DNA mini kit. The sequences were processed using Quantitative Insights into Microbial Ecology 1.9.0. Alpha-diversity was calculated using the Chao1 estimator. Beta-diversity was analyzed by UniFrac-based principal coordinates analysis (PCoA). To confirm taxa with different abundance among the groups, linear discriminant analysis effect size analysis was performed. Results: Although alpha-diversity value of the tracheostomized group was higher than that of the non-tracheostomized group, there was no statistically significant difference. In PCoA, clear separation was seen between clusters of the tracheostomized group and that of the non-tracheostomized group. In both groups, Bacteroidetes, Proteobacteria, Fusobacteria, Firmicutes, Actinobacteria were identified as dominant in phylum level. In particular, relative richness of Proteobacteria was found to be 31% more in the tracheotomized group (36.6%) than the non-tracheostomized group (5.6%)(P<0.05). In genus level, Neisseria (24%), Prevotella (17%), Streptococcus (13%), Fusobacteria (11%), Porphyromonas (7%) were identified as dominant in the tracheostomized group. In the non-tracheostomized group, Prevotella (38%), Veillonella (20%), Neisseria (9%) were genera that found to be dominant. Conclusions: It is meaningful in that the tracheostomized group has been identified a higher rate of microbiotas known as pathogenic in respiratory diseases compared to the non-tracheostomized group, confirming the possibility that the risk of opportunity infection may be higher.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.293-298
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• 2008

HCV is transmitted via various plasma derived products. Current methods to detect hepatitis C virus (HCV) are based on its antibody detection in the donated blood and plasma. Viral contamination can potentially escape such detection during the window period of infection, when no antibody is present or the level of antibody is too low to detect. It is trying to application of nucleic acid amplification tests (NAT) for the direct detection of HCV. The objective of this study was to develop a reliable NAT for the HCV RNA detection from plasma-derived products. The most useful primers was selected for NAT among 5 sets of primers. We have also found that QIAamp viral RNA isolation kit was the most efficient for HCV RNA isolation. The highest sensitivity and specificity was appeared in $48^{\circ}C$ annealing temperature and 30 pmol of primers. With a spiking of HCV to albumin, immunoglobulins and coagulation factors, NAT can detect up to 100 IU/ml. Meanwhile, COBAS amplicor HCV 2.0 afforded a lower sensitivity in high concentrated intramuscular immunoglobulins to below 500 IU/ml. Our results suggested that NAT appears to be a highly sensitive and specific method for HCV RNA detection in plasma-derived products.

• Analytical Science and Technology
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• v.21 no.4
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• pp.338-343
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• 2008

Extraction of DNA from skeletal material is of great importance in the identification of human remains, but is particularly difficult because the high amount of microbial DNA was often co-extracted with human bone DNA. We found that a phenol/chloroform extraction, followed by ultrafiltration, and cleanup by via the $QIAquick^{(R)}$ PCR purification kit yields higher amounts of human genomic DNA compared with extraction by the column affinity $method^{(R)}$ alone. Ultrafiltration extraction of human DNA from ten exhumed bone samples yielded $0.041-1.120ng/{\mu}L$ DNA (mean = $0.498ng/{\mu}L$ DNA), and purification using the column affinity resulted in $0.016-0.064ng/{\mu}L$ DNA (mean = $0.034ng/{\mu}L$ DNA). Although the STR genotyping by the column affinity method was partially successful, all DNA samples by the ultrafiltration method produced full profiles from the multiplex PCR. The efficiency of STR genotyping was in accordance with the amounts of the human DNA extracted.

• Childhood Kidney Diseases
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• v.9 no.2
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• pp.175-182
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• 2005

Purpose : Interleukin 1 receptor antagonist(IL-1ra) is an endogenous antiinflammatory agent that binds to IL-1 receptor and thus competitively inhibits the binding of IL-1$\alpha$ and IL-1$\beta$. Allele 2 in association with various autoimmune diseases has been reported. In order to evaluate the influence of IL-1ra gene VNTR polymorphism on the susceptibility to HSP and its possible association with disease severity, manifested by severe renal involvement and renal sequelae, we studied the incidence of carriage rate and allele frequency of the 2 repeats of IL-1ra allele 2($IL1RN^{*}2$) of the IL-1ra gene in children with HSP with and without renal involvement. Methods : The IL-1ra gene polymorphisms were determined in children with HSP with(n=40) or without nephritis(n=34) who had been diagnosed at Busan Paik Hospital and the control groups(n=163). Gene polymorphism was identified by PCR amplification of the genomic DNA. Results : The allelic frequency and carriage rate of $IL1RN^{*}1$ were found most frequently in patients with HSP and in controls. The allelic frequency of $IL1RN^{*}2$ was higher in patients with HSP compared to that of controls($4.7\%\;vs.\;2.5\%$, P=0.794). The carriage rate of $IL1RN^{*}2$ was higher In patients with HSP compared to that of controls($8.1\%\;vs.\;6.8\%$, P=0.916). The allelic frequency of $IL1RN^{*}2$ was higher in patients with HSP nephritis compared to that of HSP($5.3\%\;vs.\;2.9\%$, P=0.356). The carriage rate of $IL1RN^{*}2$ was higher in Patients with HSP nephritis compared to that of HSP($10.0\%\;vs.\;5.9\%$, P=0.523). Among 13 patients with heavy proteinuria(>1.0 g), 11 had $IL1RN^{*}1$, 1 had $IL1RN^{*}2$ and the others had $IL1RN^{*}4$. At the time of last follow up 4 patients had sustained proteinuria and their genotype was $IL1RN^{*}1$. Conclusion : The allelic frequency and carriage rate of $IL1RN^{*}1$ were found most frequently in patients with HSP and in controls. Our study suggests that the carriage rate and allele frequency of the 2-repeats of IL-1lra allele 2($IL1RN^{*}2$) of the IL-1ra gene may not be associated with susceptibility and severity of renal involvement in children with HSP (J Korean Soc Pediatr Nephrol 2005;9:175-182)