• Title/Summary/Keyword: ITS-PCR

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Evaluation of Xenotropic Murine Leukemia Virus and its R426Q Polymorphism in Patients with Prostate Cancer in Kerman, Southeast of Iran

  • Reza, Malekpour Afshar;Fahimeh, Gadari;Reza, Mollaie Hamid
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.8
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    • pp.3669-3673
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    • 2012
  • A role for the xenotropic murine leukemia virus (XMRV) in prostate cancer development has been postulated. To answer questions regarding the prevalence of XMRV in Iranian patients with prostate cancer and its association with the RNASEL R462Q polymorphism, we here investigated a series of cases in Kerman, in the Southeast of Iran, and sought to verify the association with the R462Q using Real Time PCR Method. Prostate tissue specimens of 200 patients with prostate cancer were genotyped for R462Q by real time polymerase chain reaction allelic discrimination and were screened for XMRV proviral DNA by real time polymerase chain reaction specific for the envelope gene. Of 200 patients in this study 8 (4%) cases were positive for XMRV, the QQ allele being the most frequenct regarding the R426Q polymorphism while in negative patients it was the RQ allele. There was significant correlation between high pathological scores and XMRV positive samples. No significant relationship was found between age groups and XMRV results. XMRV was only found in patients with QQ and RQ alleles, not RR. XMRV is detectable in tumor prostate tissue from some patients with prostate cancer, independent of R462Q.

Efficient Transformation of Trifolium repens L. Using Acetosyringone (Acetosyringone을 이용한 효율적인 White Clover의 형질전환)

  • TaeHoKwon
    • Korean Journal of Plant Resources
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    • v.10 no.2
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    • pp.107-113
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    • 1997
  • Transformants of White Clover(Trifolium repens L.) were efficiently produced from immature seed derived callus cocultivated with Agrobacterium twnefaciens LBA4404 harboring plant binary vector. pBI121, using acetosyringone. The mean frequencies of transformants on the two kanamycin-containing media were 16 to 19% when the immature seed-derived calli were infected with bacteria cultured in the presence of 100$\mu$M acetosyringone compared with 7% in media without acetosyringone. Transgenic white clover was subject to molecular analysis for integration into plant nuclear genome and expression of $\beta$-glucuronidase(GUS) gene. PCR and Northern blot analyses demonstrated that GUS gene was integrated into white clover nuclear genome and expressed into its mRNA. The expression of GUS gene into its protein was confirmed by spectrophotometric assay of GUS activity.

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Genotype Analyses of Methicillin Resistant Staphylococcus aureus Isolated from Intensive Care Units (중환자실에서 분리된 Methicillin 내성 Staphylococcus aureus의 유전자형 분석)

  • Kim, Yun-Kyung;Gwak, Mi-Kyung;Lee, Ji-Min;Hong, Hae-Sook
    • Journal of Korean Biological Nursing Science
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    • v.10 no.2
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    • pp.147-153
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    • 2008
  • Purpose: Methicillin Resistant Staphylococcus aureus (MRSA) has become a major clinical problem and one of the major nosocomial pathogen worldwide. The aim of the study was to investigate the epidemiological characteristics of genotypes of MRSA isolated in the A-hospital ICU. Methods: In the period between December 2007 and May 2008, MRSA was isolated from ICU patients and its surrounding environment. Polymerase Chain Reaction (PCR) was conducted for the detection of MRSA gene. The incidence of MRSA in the clinical isolates of Staphylococcus aureus was examined by using a multiplex PCR. The spa gene of Staphylococcus aureus encodes protein A and is used for typing of MRSA. We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a hospital. Results: Two different genotypes of MRSA were identified with 90 isolated from the patients and its surrounding environments in the ICU. Conclusion: This study may contribute to the development of effective strategies for preventing nosocomial infections. Genotyping may have more general application for the study of MRSA epidemic outbreak in hospital and community infection.

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MiR-150-5p Suppresses Colorectal Cancer Cell Migration and Invasion through Targeting MUC4

  • Wang, Wei-Hua;Chen, Jie;Zhao, Feng;Zhang, Bu-Rong;Yu, Hong-Sheng;Jin, Hai-Ying;Dai, Jin-Hua
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.15
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    • pp.6269-6273
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    • 2014
  • Growing evidence suggests that miR-150-5p has an important role in regulating genesis of various types of cancer. However, the roles and the underlying mechanisms of miR-150-5p in development of colorectal cancer (CRC) remain largely unknown. Transwell chambers were used to analyze effects on cell migration and invasion by miR-150-5p. Quantitative real-time PCR (qRT-PCR), Western blotting and dual-luciferase 3' UTR reporter assay were carried out to identify the target genes of miR-150-5p. In our research, miR-150-5p suppressed CRC cell migration and invasion, and MUC4 was identified as a direct target gene. Its effects were partly blocked by re-expression of MUC4. In conclusiomn, miR-150-5p may suppress CRC metastasis through directly targeting MUC4, highlighting its potential as a novel agent for the treatment of CRC metastasis.

Molecular Analysis of Anisakis Type I Larvae in Marine Fish from Three Different Sea Areas in Korea

  • Sohn, Woon-Mok;Kang, Jung-Mi;Na, Byoung-Kuk
    • Parasites, Hosts and Diseases
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    • v.52 no.4
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    • pp.383-389
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    • 2014
  • Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA cox1 revealed that the larvae showed interesting distribution patterns depending on fish species and geographical locations. Anisakis pegreffii was predominant in fish from the Yellow Sea and the South Sea. Meanwhile, both A. pegreffii and A. simplex sensu stricto (A. simplex s.str.) larvae were identified in fish from the East Sea, depending on fish species infected. These results suggested that A. pegreffii was primarily distributed in a diverse species of fish in 3 sea areas around Korea, but A. simplex s.str. was dominantly identified in Oncorhynchus spp. in the East Sea.

Development of a Virus Concentration Method and its Application for the Detection of Noroviruses in Drinking Water in China

  • Liu, Junyi;Wu, Qingping;Kou, Xiaoxia
    • Journal of Microbiology
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    • v.45 no.1
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    • pp.48-52
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    • 2007
  • A new procedure for the concentration of nonoviruses from water samples has been developed. This procedure (calcium flocculation-citrate dissolution method) uses the following steps: virus flocculation formed by treatment with 1 M $CaCl_2$ and 1 M $Na_2HPO_4$, virus release by sodium citrate dissolution (0.3 M Na citrate, pH 3.5), and virus re-concentration by ultrafiltration. When reverse transcription (RT)-PCR was performed after the procedure, the overall detection sensitivity for seeded noroviruses in a one liter drinking water sample was as low as 1 RT-PCR unit, which is equal to a $10^{-6}$ dilution of the virus sample. This approach showed at least a 5-fold-higher sensitivity than the current method with its three steps of adsorption-elution-concentration. The newly developed procedure was used to test different brands of bottled drinking water from China for putative contamination with noroviruses. A total of 144 samples were analyzed; all of the samples were negative for norovirus specific nucleic acids.

Screening of Antioxidative Activity of Bifidobacterium Species Isolated from Korean Infant Feces and Their Identification

  • Kim, Ji-Youn;Park, Soo-Im;Heo, Tae-Ryeon
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.3
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    • pp.199-204
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    • 2003
  • Among 59 Korean isolated, 20 were confirmed as members of the genus Bifidobaferium species based on gram staining, microscopic examination of cell morphology and the TLC method, The oxygen tolerance and antioxidative activities of these 20 Bifidobacterium strains and 5 standard Bifidobaferium strains were tested. All the strains demonstrated antioxidative activities as regards inhibiting linoleic acid peroxidation. The antioxidative activities of isolated and standard strains were found to range from 10.7-46.4% and from 10.7-22.2%, respectively. In addition, all tested strains exhibited a Scavenging ability on DPPH free radicals, range from 15-41% for the isolated strains and 8.3-22% for the standard strain. Accordingly. the isolated Bifidobarterium strains demonstrated higher antioxidative artivities than the 5 standa rd Bifidobarterium strains. On the base of grades for each test, HJL 7511 was identified 35 the best strain, followed by HJL 7501. 2 strains were identified with Polymerase Chain Reaction (PCR) assay using group-specific primers designed from the nucleotide Sequences of the 16S rRNA and internal transcribed spacer (ITS) regions of the Bifidobacteria. Based on the Sequencing results, HJL 7511 and HJL 7501 were identified as Bifidobacterium infantis.

Anti-allergic Effects of Shinbijen (신비전(新鼻煎)의 항알레르기 효과)

  • Kim, Kyung-Yeol;Kim, Sang-Tae;Lee, Jong-Hwa
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.21 no.6
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    • pp.1525-1533
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    • 2007
  • The discovery of drugs on the treatment of rhinitis and allergic disease is a very important subject in human heath. The Shinbijen has been used for centruries as a traditional medicine in Korea and is known to have an anti-inflammatory effect. However, its specific mechanism of action is still unknown. In this report, we investigated the effect of hot water extract from Shinbijen on OVA-mediated allergic reaction and studied its possible mechanism of action using fluorescenc microscopy and RT-PCR analysis. Shinbijen inhibited OVA-induced rhinitis, total cells in BFA and lymphocyte related to inflammation in mice. Shinbijen decreased immuno response, which activated by IL-4, COX-2 and iNOS expression. in tissue. Shinbijen reduced inflammatory molecule release from mice lung tissue and CD4/CD8 cells activated by cardiac blood. Shinbijen decreased OVA-induced IL-4 and iNOS levels in pulmonary alveoli. Our findings provide evidence that Shinbijen inhibits OVA-induced allergic reactions, and also demonstrate the involvement of inflammation and allergic disease in these effects.

Development of Molecular Marker to Detect Citrus Melanose Caused by Diaporthe citri from Citrus Melanose-like Symptoms

  • Choi, Cheol-Woo;Jung, Kyung-Eun;Kim, Min-Ju;Yoon, Su-Hyeon;Park, Suk-Man;Jin, Seong-Beom;Hyun, Jae-Wook
    • The Plant Pathology Journal
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    • v.37 no.6
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    • pp.681-686
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    • 2021
  • It is difficult to distinguish melanose and melanoses-like symptoms with the naked eye because they appear similar. To accurately detect melanose symptoms caused by Diaporthe citri from melanose-like symptoms, we developed PCR-based specific primers Dcitri by aligning the internal transcribed spacer (ITS) region of D. citri with the ITS of Diaporthe cytosporella, Diaporthe foeniculina, Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria citri, and Fusarium oxysporum found on citrus peel. PCR results showed that the specific product was amplified in D. citri but not in other isolates including, C. gloeosporioides, B. cinerea, A. citri, F. oxysporum. In addition, specific products were observed in melanose symptoms caused by D. citri but not in melanose-like symptoms, such as copper-injury, sunscald, damages by yellow tea thrips, and pink citrus rust mite. Using the Dcitri primers developed in this study, it is expected that melanose caused by D. citri could be accurately distinguished from melanose-like symptoms.

Validation of Stem-loop RT-qPCR Method on the Pharmacokinetic Analysis of siRNA Therapeutics (Stem-loop RT-qPCR 분석법을 이용한 siRNA 치료제의 생체시료 분석법 검증 및 약물 동태학적 분석)

  • Kim, Hye Jeong;Kim, Taek Min;Kim, Hong Joong;Jung, Hun Soon;Lee, Seung Ho
    • Journal of Life Science
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    • v.29 no.6
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    • pp.653-661
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    • 2019
  • The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.