Comparison of Swabbing Solution Volume and gDNA Extraction Kits on DNA Recovery from Rigid Surface.

For bacteria sampling studies, various collection methods have been used to identify bacteria. To obtain accurate information about bacteria, high quality samples should be obtained. In order to obtain a high quality sample, a stable and large number of DNA copies must be collected. This study compared the efficiency of different methods of bacterial gDNA extraction and bacteria collection according to swabbing solution volumes and types. The efficiency of bacterial genomic DNA extraction was compared using a AccuPrep® Genomic DNA Extraction kit, a QIAamp® DNA Mini kit, and a MOBIO® DNeasy PowerSoil kit. The DNA Mini kit was shown to extract the highest amount of gDNA, and sub-experiments were conducted using this kit. Phosphate-buffered saline and phosphate-buffered saline with 0.1% Tween 20 were used as collection solutions of various volumes (0, 40, 50, 60, 70, 80, 90, 100, 110, and 120 µL) using cotton swabs. Bacteria collection efficiency was highest when 70 µL PBS was used. The target strains collected in this experiment were Staphylococcus aureus and Escherichia coli, and these were quantified using the number of colony-forming units, DNA concentrations, and the number of DNA copies. These results can be used to efficiently bacterial collection for experiments in various fields.

Development of a novel DsRed-NLS vector with a monopartite classical nuclear localization signal.

The nuclear localization signal (NLS) marks proteins for transport to the nucleus and is used in various applications in many fields. NLSs are used to achieve efficient and stable transport of biomolecules. Previously, commercial vectors used in NLS studies contained three iterations of the NLS sequence, but these sequences can affect experimental results and alter protein function. Here, we investigated a new vector using a single classical NLS sequence with a mutation in pDsRed2-C1-wt to reduce experimental artifacts. In the newly constructed pDsRed2-C1-1NLS vector, the NLS sequence is placed near the multiple cloning sites of pDsRed2-C1-wt, and the multiple cloning site region was designed to facilitate insertion of the desired gene by site-directed mutagenesis. Fluorescent protein expression in the nucleus can be visually confirmed. The results show that the fluorescent protein was bound to the transport protein. The constructed vector had a cell survival rate of 89-95% and a transfection efficiency of 39-56% when introduced into animal cells, which are similar to those of other NLS vectors. Additionally, the constructed NLS vector can be used to demonstrate complementary binding between target proteins, and that the target protein is transported by the NLS transport system. Especially, we show that the vector can be useful for experiments involving the S100A10 gene. In addition, the constructed vector is useful for studies of genes and proteins that show potential for gene therapy or drug delivery applications.

Influence of swabbing solution and swab type on DNA recovery from rigid environmental surfaces.

Determination of the metagenome has become an important component of forensic identification, which requires efficient environmental sampling techniques. Therefore, in this study, we compared the efficiency of sample collection using swabbing with cotton swabs and three types of medical swabs (S7, S22, S24) along with three different solutions: phosphate-buffered saline (PBS), 1% Tween 20 + 1% glycerol in PBS (TG), and GS commercial solution (Noble Bio, Hwaseong, Republic of Korea). Combinations of the three solutions with the three types of swabs were tested at different volumes (cotton swab, S7: 0, 30, 50, 70 µL; S22, S24: 0, 70, 100, 130 µL). Escherichia coli and Staphylococcus aureus were selected as representative environmental microbial samples, and the number of colony-forming units (CFUs), DNA concentration, and DNA copy numbers were compared across groups. The sampling process had a clear effect on the efficiency of extraction, which allowed for determination of a more efficient sample sampling method. In particular, cotton swabs showed 2-10-fold greater CFUs of both species than the medical swabs, and resulted in significantly greater amounts of extracted DNA. TG was found to be the most efficient solution for bacterial DNA extraction, with higher CFUs and DNA obtained than with the other three solutions at all volumes tested. This study highlights the need for a standardized sampling method that can be applied to all environmental samples, especially for microbial quantification, and provides valuable reference data for the efficient collection of environmental samples for metagenomic analyses in microbial-based forensic assessments.

Changes in Gene Expression in Human Epithelial and Mast Cells in Response to Vesicles from Klebsiella pneumonia ATCC 13883.

We investigated alterations in the expression of immune-related genes in epithelial cells and mast cells treated with outer membrane vesicles (OMVs) derived from Klebsiella pneumoniae (KpOMVs). Previous studies have shown that OMVs contain substances that enable their delivery to host cells and induce an immune response. Our results indicate an increase in expression of genes such as IL-8, IL-1b, MIP-1α, HMOX1, HSPA1A, and IL-24 in epithelial cells and mast cells treated with KpOMVs. The pathogenicity of KpOMVs was confirmed by measuring the changes in the expression of these immune-related genes.

Trends of the Species and Antimicrobial Susceptibility of Microorganisms Isolated from Blood Cultures of Patients / 대한임상미생물학회지

BACKGROUND: Blood culture is an important procedure for the determination of the etiologic agent of septicemia. Analysis of the blood culture results can provide clinicians with very important information for the empirical treatment of patients. METHODS: In this study the blood cuture results at Chosun University Hospital during the years 2002 to 2005 were analysed to determine the species and antimicrobial susceptibility of the isolates. Blood culture bottles were incubated in BACTEC 9240 blood culture system; the isolates were identified by Vitek II, and antimicrobial susceptibility was tested by Vitek II system or the NCCLS disk diffusion method. RESULTS: Positive blood cultures were obtained from 1,520 (18.5%) patients. Among the microorganisms isolated from blood culture, 97.0% were aerobic and facultative anaerobic bacteria and 2.8% were fungi. Frequently isolated organisms in decreasing order were coagulase-negative staphylococci (CNS), Escherichia coli, Staphylococus aureus, Stenotrophomonas maltophilia, Serratia marcescens, and Klebsiella pneumoniae. The proportion of Pseudomonas aeruginosa isolates resistant to ceftazidime and imipenem was increased during the study period. CONCLUSION: E. coli was the most frequent etiologic agent of bacteremia except CNS, common contaminants of skin, at Chosun University Hospital. It seems to be necessary to enhance infection control measures to cope with an increasing number of the resistant bacteria to various antibiotics.

The Effect of Tai Chi Movement in Patients with Rheumatoid Arthritis / 간호학회지

PURPOSE: This study was performed to verify the effect of Tai Chi exercise on patients with rheumatoid arthritis particularly their level of pain, fatigue, sense of balance and daily life performance (ADL). METHOD: It employed a non-equivalent control group pre- and post-test design. The research instruments used in this study were pain, fatigue, sense of balance and ADL. Thirty-two patients in the experimental group carried out 50 minutes of Tai Chi exercise for 12 weeks, and 29 patients in the control group did not. Before and after the experiment, both groups were tested for pain, fatigue, sense of balance and ADL. Collected data were processed using the SPSS/WIN 10.0 program analyzed by the frequency, percentage, X2-test, and t-test. RESULTS: Pain and fatigue significantly decreased in the experimental group. However the improvement in ADL of the rheumatoid arthritis patients was not statistically significant but their sense of balance was enhanced significantly. CONCLUSION: Tai Chi exercise is an effective nursing intervention that can be used for rheumatoid arthritis patients.

Comparison of Clinical Characteristics of Patients with Rotavirus Gastroenteritis Relative to the Infecting Rotavirus G-P Genotype / 대한진단검사의학회지

BACKGROUND: Group A rotavirus is a major cause of severe diarrhea in children throughout the world. For the proper management of rotavirus infections, it will be helpful to know their clinical characteristics according to the G and P genotypes of the infecting virus. METHODS: The diarrheal stool specimens from patients hospitalized in Chosun University Hospital during 2002-2003 were tested for rotavirus by Dipstick 'Eiken' Rota kit. Rotavirus antigen-positive stool specimens were analyzed for group A rotavirus by RT-PCR, and the group A-positive PCR products were genotyped for P and G types by PCR. RESULTS: Among the 119 specimens analyzed for genotypes, the predominant strain was genotype G4P[6] (51.3%), followed by G2P[4] (19.3%), G1P[8] (7.6%), G3P[8] (5.0%), and G9P[8] (4.2%). To examine the characteristics of each rotavirus genotype, a clinico-epidemiological study was performed for 100 patients whose medical records were available. The frequencies of diarrhea, vomiting, dehydration, and fever; the rates of nosocomial infection and transfer from other hospitals; and the mean severity scores were significantly different among the patients infected with different types of rotavirus. Especially, patients with G4P[6] type were more likely than those infected with other genotypes to show the following distinct features: Most patients showed milder symptoms and were neonates transferred from other obstetric hospitals and 68.4% of the cases were nosocomial infection. G4P[6] strains were isolated almost all along the year. The mean severity scores of patients infected by G4P[6], G2P[4], G1P[8], G3P[8], and G9P[8] strains were 6.8, 9.5, 8.0, 9.0, and 10.8, respectively. CONCLUSIONS: Many features of rotavirus infections including the epidemic period, rate of nosocomial infection, age and severity of symptoms were different according to the genotypes of the infecting virus.

Evaluation of the detectability of Vitek II System for Inducible Clindamycin Resistance in Staphylococci / 대한진단검사의학회지

BACKGROUND: While broth based antimicrobial susceptibility test methods work well for the detection of the majority of antimicrobial resistance mechanisms, antimicrobial resistance mechanism in some microorganisms may not be detected by these methods. The purpose of this study was to compare Vitek II system with a standard method for the ability to detect inducible clindamycin resistance in Staphylococcus aureus. METHODS: Of 200 clinical isolates of S. aureus tested, 183 were methicillin resistant (MRSA) and 17 were methicillin susceptible (MSSA). A disk approximation test (Clinical Laboratory Standards Institute; CLSI, Wayne, PA, USA) was performed as the standard method by placing standard erythromycin and clindamycin disks in adjacent positions. Vitek II ID-GPI (bioMerieux, Durham, NC, USA) was used for identification and Vitek AST-P536 (bioMerieux, Durham, NC, USA) for antimicrobial susceptibility tests. RESULTS: Clindamycin resistance rates of S. aureus tested by disk diffusion and Vitek II system were 89% and 56%, respectively. All but one inducible clindamycin resistant MRSA isolates were susceptible to clindamycin by Vitek II system. Five inducible clindmycin resistant MSSA isolates were all susceptible to clindamycin by Vitek II system. Vitek II system did not detect the inducible clindamycin resistance in S. aureus. CONCLUSIONS: Our results showed that Vitek II system was unacceptable for the detection of inducible clindamycin resistance in S. aureus. We suggests that the disk approximation test should be used to detect the inducible clindamycin resistance in S. aureus.

Usefulness of Serum Cystatin C for the Evaluation of Renal Function in Diabetic Patient / 대한진단검사의학회지

BACKGROUND: Diabetic nephropathy is the most frequent complication in patients with diabetes mellitus (DM). In clinical practice, the glomerular filtration rate (GFR) is often estimated from serum creatinine. Recently, serum cystatin C has been suggested being a better parameter for diagnosis of impaired renal function. We evaluated serum cystatin C as a potential new marker of GFR in diabetes patients. METHODS: Serum cystatin C and serum creatinine (sCr) were measured in 73 DM patients to evaluate their usefulness in diabetic patients. DM patients were divided into three groups (whole DM patients, albuminuric patients, and DM patients with sCr<1 mg/dL). Serum cystatin C and sCr were compared with creatinine clearance (CCr). RESULTS: The overall correlation coefficient for the reciprocal of serum cystatin C was superior to that of the reciprocal of serum creatinine in all three patient groups. With CCr cut-off values of 60 mL/min and 80 mL/min, receiver operating characteristic (ROC) plotting demonstrated that serum cystatin C had a higher sensitivity and specificity for detecting decreased GFR than did serum creatinine in all three patient groups. CONCLUSIONS: These findings suggest that serum cystatin C is superior to serum creatinine as a marker of GFR measured by correction or mean ROC-plot AUC in diabetic patients; therefore, serum cystatin C could be used for the early detection of the impairment of renal function.

The Polymerase Chain Reaction Applying dUTP-UDG Protocol for Detection of Mycobacterium tuberculosis / 대한임상병리학회지

BACKGROUND: The polymerase chain reaction(PCR) assay is rapid, sensitive analytical technique but has problem of high false-positive rate. We applied dUTP-UDG PCR (dU-PCR) method to prevent carryover contamination major source of high false positive in PCR assays, for detection of Mycobacterium tuberculosis. METHODS: The PCRs for detection of M. tuberculosis were performed with P1 and P2 primers based on IS6110 repeated sequence. FTC-2000 was used for capillary PCR and Uno-Thermoblock was used for heating block PCR. In order to evaluate the effect of dU-PCR controlling carryover contamination, PCRs were performed in the presence of UDG and the absence of UDG. To compare the sensitivity of usual dT-PCR with dU-PCR, chromosomal DNA of M. tuberculosis ranging 500pg to 0.5fg were amplified by dT-PCR and dU-PCR method using two different thermocycler, capillary and heating block type, respectively. RESULT: The dU-PCR using UDG prevented carryover contamination by amplicon DNA up to 500pg. By capillay PCR method, the lower limits of detectability of dT-PCR and dU-PCR were 0.5fg and 500fg, respectively, which indicates the sensitivity of dU-PCR was lower than dT-PCR. But by heating block method, the lower limits of detectability of both method of dU and dT-PCR were 0.5fg. So the sensitivity of dU-PCR was same as dT-PCR. CONCLUSION: The dU-PCR by heating-block method was sensitive test for detection of M. tuberculosis that effectively prevent carryover contamination by amplicon.

Comparison of DNA Extraction Methods for the Polymerase Chain Reaction of Mycobacterium tuberculosis / 대한임상병리학회지

No abstract available.