• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1616-1621
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• 2007

We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was $1{\times}10^4\;CFU/g$ feces for S. flexneri and $2{\times}10^4\;CFU/g$ feces for S. typhimurium; this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.

• BMB Reports
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• v.40 no.3
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• pp.444-447
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• 2007

Polymerase chain reaction (PCR) is a powerful technique in molecular biology and is widely used in various fields. By amplifying DNA fragments, PCR has facilitated gene cloning procedures, as well as molecular genotyping. However, the extraction of DNA from samples often acts as a limiting step of these reactions. In particular, the extraction of PCR-compatible genomic DNA from higher plants requires complicated processes and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors, including polyphenolic compounds. We recently developed a novel solution, referred to as AnyDirect, which can amplify target DNA fragments directly from whole blood without the need for DNA extraction. Here, we developed a simple lysis system that could produce an appropriate template for direct PCR with AnyDirect PCR buffer, making possible the direct amplification of DNA fragments from plant leaves. Thus, our experimental procedure provides a simple, convenient, non-hazardous, inexpensive, and rapid process for the amplification of DNA from plant tissue.

• Journal of Microbiology and Biotechnology
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• v.21 no.6
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• pp.659-664
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• 2011

Staphylococcus aureus (SA) bacteremia is associated with high mortality, and often results in metastatic infections. The methicillin-resistant SA (MRSA) is an urgent health care issue, as nosocomial infections with these bacteria represent limited treatment alternatives. Samples of whole blood containing challenge inoculums of SA and MRSA strains were passed through columns packed with surfaceheparinized polyethylene beads. The bound bacteria were eluted and quantitatively determined by culturing and by real-time PCR. Significant amounts of both SA and MRSA adhered to the heparinized beads (more than 65% of inoculated bacteria). After rinsing with buffer at high ionic strength, viable bacteria or bacterial DNA were eluted from the columns, indicating that the binding was specific. The conclusions that can be made from these experiments are that, as earlier reported in the literature, the high affinity of SA to heparin is retained in whole blood, and MRSA in whole blood binds to heparin with similar or higher affinity than SA. It should be possible to lower the amount of SA and/or MRSA from the blood of infected patients to levels that could be taken care of by the immune system. In previous studies, we have shown that passing blood from septic patients over beads coated with end-point-attached, biologically active heparin is a useful technique for regulating the levels of heparinbinding cytokine. These findings in combination with the present findings indicate the possibility of creating an apheresis technology for treatment of sepsis caused by SA and/or MRSA.

• Journal of Genetic Medicine
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• v.9 no.1
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• pp.11-16
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• 2012

Purpose: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. Materials and Methods: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. Results: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. Conclusion: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.11
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• pp.1689-1693
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• 2007

The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing goat embryos. Based on the amelogenin gene located on the conservation region of X- and Y- chromosomes, a pair of primers was utilized and the system of PCR was established to amplify a 262 bp fragment from the X- chromosome in female goats, and a 262 bp fragment from X- chromosome and 202 bp fragment from the Y- chromosome in male goats, respectively. The accuracy and specificity of the primers were assessed using DNA template extracted from goat whole blood sample of known sex. 100% (10/10) concordance was obtained by using the PCR assay. Fifty-one biopsied embryos were transferred into 25 recipient goats on the same day that the embryos were collected and sex of the kid was confirmed after parturition. Eighteen kids of predicted sex were born. The biopsied samples from 51 goat embryos were amplified with 100% efficiency and 94.7% accuracy. In conclusion, our results indicated that PCR sexing protocols based on the amelogenin gene is highly reliable and suitable for sex determination of goats.

• Journal of Veterinary Clinics
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• v.20 no.4
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• pp.478-481
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• 2003

Canine juvenile cellulitis (CJC) is a well-recognized lymphocutaneous disease that is seen in young dogs. CJC seemed to be immunologic disorder and may have a hereditary aspect. Exact pathogenesis and cytokine regulation on the immune system of CJC are not clear. CJC was diagnosed in two puppies hospitalized in Veterinary Teaching Hospital of Chungbuk National University. To investigate the cytokine regulation on CJC, RT-PCR was performed with CJC affected dogs. RT-PCR 1 was performed with whole blood sample (CJC-B) and fine needle aspirates of the inguinal lymph node (CJC-LN) from case 1-dog, which included $TNF-\alpha,$ $IL-1\beta,$ $IFN-\gamma,$ IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 and $\beta-actin.$ Blood sample from a normal dog (N-B) served for a negative control of RT-PCR 1 (case 1). $IFN-\gamma,$ IL-2, IL-4, IL-5, IL-8, IL-10 and IL-12 transcripts were not expressed in all sample. $TNF-\alpha$ and $IL-1\beta,$ were not transcripted from CJC-B but from CJC-LN. On RT-PCR 2 (case 2), submandibular lymph node aspirates were used and $TNF-\alpha,$ IL-10, $IFN-\gamma$ and $IL-1\beta$ were expressed. $TNF-\alpha,$ 1L-10 and $IFN-\gamma$ were secreted from activated macrophages enhance the inflammation in tissue. These results imply that abnormally increased macrophages secret $TNF-\alpha$ and $IL-1\beta$ in the affected lymph nodes, which attract neutrophils and cause inflammation in CJC.

• Parasites, Hosts and Diseases
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• v.55 no.5
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• pp.561-564
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• 2017

This report describes a dog infected with Hepatozoon canis, the first canine infection in the Republic of Korea. A 2-year-old intact male Maltese dog presented with anorexia and depression. Physical examinations revealed mild dehydration and hyperthermia ($39.8^{\circ}C$), and blood analysis showed pancytopenia. Diff-Quik staining of blood smear specimens showed the presence of ellipsoidal shaped structures (gamonts of H. canis) within a small number of neutrophils. Real-time PCR analysis using whole blood confirmed infection by H. canis. The clinical condition of the dog improved after symptomatic treatment and administration of doxycycline. Although a molecular epidemiologic survey in Korea showed H. canis infection of dogs, to our knowledge this is the first report of a dog infection in Korea molecularly shown to be H. canis.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.189-195
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• 2006

Behcet's disease (BD) is a chronic, multisystemic disorder which is more frequently seen in the Mediterranean basin, Middle East, and Far East. The causes and pathogenesis of BD are unknown although many possibilities are being investigated. The diagnosis of BD is based on clinical manifestations because there are no pathognomonic laboratory tests. So, the purpose of this study was to examine the prevalence of HLA-B51 in patients with BD. We used the whole blood of 33 patients diagnosed with BD at Chosun University Hospital from August 2003 to January 2006. For the HLA-B51 test, we extracted the DNA from the whole blood of 33 BD patients, and we investigated it through the nested PCR method. Data were analyzed using the SPSS/PC 10.0. The frequencies of gender of the 33 cases diagnosed as BD were male 13 (39.4%) and female 20 (60.6%). The frequencies of age group of the 33 cases diagnosed as BD were 20 yrs 8 (24.2%), 30 yrs 12 (36.4%), 40 yrs 8 (24.2%), 50 yrs 1 (3.0%), and 60 yrs and 70 yrs 2 (6.1%), respectively. The frequencies of HLA-B51 of the 33 cases diagnosed as BD were HLA-B51-negative 18 (54.5%) and HLA-B51-positive 15 (45.5%). In conclusion, BD occurred more often in women than men (1: 1.53), and the mean age of the BD patients was 39.8 years old. HLA-B51 was positive in 45.5% of patients with BD, and was statistically significant in age (p<0.05).

• Biomedical Science Letters
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• v.27 no.4
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• pp.216-222
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• 2021

Colorectal cancer (CRC) is major cancer with high incidence and mortality worldwide. It is known that most CRCs arise from precursor adenomatous polyps (APs). Recently, microRNA (miRNA) has been proposed as a biomarker for various cancers including CRC. In this study, the expression patterns of miR-29a in the whole blood (WB) of CRC, AP, and control groups were analyzed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to evaluate the expression level of miR-29a in patients with colorectal neoplasm (CRN) including CRC and AP. As a result, the relative expression of miR-29a was significantly decreased in the patients with CRN compared to the control group (P<0.001). The results were in agreement with previous in vitro cell studies and studies that used tissue and feces samples, suggesting that miR-29a in WB may be useful in demonstrating the status of colorectal tissue. Additionally, we divided the control group into healthy control (HC) without any colorectal symptoms and non-tumor control (NTC) with colorectal symptoms but without any CRN. And then the relative expression of miR-29a was also significantly decreased in the NTC group compared to the HC group (P<0.001). Therefore, our study revealed that miR-29a can differentiate patients with CRN from HC group, but they are also involved in the early stage of inflammatory response and cannot be specific biomarkers for CRN.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.7
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• pp.2923-2928
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• 2015

Background: Gallbladder carcinoma (GBC) has been stated as an Indian disease, with the highest number of cases being reported from certain districts of northeast India, which has an ethnically distinct population. Unfortunately there are no scientific reports on the underlying molecular mechanisms associated with the pathogenesis of the disease from this region. Aim: The present study evaluated the role of differential expression of human telomerase reverse transcriptase (hTERT) in the development of gall bladder anomalies. Materials and Methods: Blood and tissue samples were collected from patients undergoing routine surgical resection for clinically proven cases of gallbladder disease {cholelithiasis (CL, n=50), cholecystitis (CS, n=40) and GBC (n=30) along with adjacent histopathologically proved non-neoplastic controls (n=15)} with informed consent. Whole blood was also collected from age and sex matched healthy controls (n=25) for comparative analysis. Differential hTERT mRNA expression was evaluated by semi-quantitative rt-PCR and real-time PCR based analysis using ${\beta}$-actin as an internal control. Evaluation of differential hTERT protein expression was studied by Western blot analysis and immunoflourescence. Statistical analysis for differential expression and co-relation was performed by SPSSv13.0 software. Results: Gallbladder anomalies were mostly prevalent in females. The hTERT mRNA and protein expression increased gradiently from normal