• Title/Summary/Keyword: Competitive PCR

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Endometrium from Women with Endometriosis Expresses Decreased Levels of Plasminogen Activator Inhibitor-1 and Tissue Inhibitor of Metalloproteinase-3 Compared to Normal Endometrium (자궁내막증 환자와 정상 여성의 자궁내막에서 TIMP-3와 PAI-1 mRNA 발현 차이에 관한 연구)

  • 정혜원
    • Development and Reproduction
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    • v.3 no.1
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    • pp.29-38
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    • 1999

  • The pathogenesis of endometriosis is unknown, but retrograde menstruation is widely accepted as an etiology. Refluxed endometrium from endometriosis patients is more prone to implant and invade peritoneum possibly through the action of extracellular proteolysis. This proteolytic action may involve plasminogen activators and the collagenase system. Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) play a critical role in the breakdown of extracellular matrix components and basement membrane in the processes of implantation and tumor invasion. PAs are inhibited by plasminogen activator inhibitor (PAI) and MMPs activity is inhibited by tissue inhibitor of metalloproteinase (TIMP). To test the hypothesis that lower expression of PAI-1 and TIMP-3 in endometrium from women with endometriosis, we investigated their PAI-1 and TIMP-3 expression by quantitative competitive RT PCR in endometrium from women with and without endometriosis. Endometrial tissues were obtained from 14 patients with severe endometriosis and 14 patients without endometriosis. Total RNA was extracted and reverse transcribed into cDNA, and quantitative competitive PCR (QC PCR) was performed to evaluate PAI-1 and TIMP-3 mRNA expression. Endometrium from patients with endometriosis showed decreased expression of PAI-1 and TIMP-3 mRNA compared to endometrium from control in luteal phase (p<0.05). Our results suggest that endometrium from women with endometriosis expresses lower levels of PAI-1 and TIMP-3 than endometrium from normal women. Endometrium from endometriosis patients may be more invasive and prone to peritoneal implantation than control because of higher PA and MMP enzymatic activity. Thus, increased proteolytic activity may be one of the reasons for the invasive properties of the endometrium resulting in the development of endometriosis.

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Primer for the Potato Specific Internal Control DNA and Screening Method for the Genetically Modified Potatoes by Competitive Duplex-PCR (감자 특이 Internal Control DNA 증폭용 Primer와 이를 이용한 유전자 변형 감자의 경쟁적 이중 PCR 검정법)

  • Seo, Hyo-Won;Yi, Jung-Yoon;Cho, Hyun-Mook;Kim, Sung-Yeul
    • Journal of Plant Biotechnology
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    • v.29 no.4
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    • pp.235-240
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    • 2002

  • We report the new method for the screening of genetically modified potato by competitive duplex-PCR using the potato specific single oligomer primer for the internal control and CaMV 35S promoter or NOS terminator specific primers. The single oligomer primer (rAGU4A) amplify the potato specific internal control band from the homozygous potato genomic DNA in the RAPD profiles of all analyzed potato varieties. The 530 bp internal control DNA was amplified independently to CaMV 35S promoter or NOS terminator DNA and identified as repetitive or microsatellite DNA of potato (AF541972). With this new technique, the transgenic potatoes which were transformed with vectors contained the different foreign genes are analyzed. In case of the commercialized transgenic potato varieties, 'Hew Leafs', those two genetic factors are used for promoter and terminator respectively So, this new PCR technique should be a promising method of cost effective and accurate screening for the commercialized GM potatoes on market.

  • https://doi.org/10.5010/JPB.2002.29.4.235 인용 PDF KSCI

Estimation of Distribution of a Commensal Thermophile in Soil by Competitive Quantitative PCR and Terminal Restriction Fragment Length Polymorphism Analysis

  • Rhee, Sung-Keun;Hong, Seung-Pyo;Bae, Jin-Woo;Jeon, Che-Ok;Lee, Seung-Goo;Song, Jae-Jun;Poo, Ha-Ryoung;Sung, Moon-Hee
    • Journal of Microbiology and Biotechnology
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    • v.11 no.6
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    • pp.940-945
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    • 2001

  • Symbiobacterium toebii has been previously reported as a novel commensal thermophile exhibiting a commensal interaction with thermophilic Geobacillus sp. SK-1. We investigated the distribution of this commensal thermophile in various soils using molecular methods, such as quantitative PCR and terminal restriction fragment polymorphism analysis. Based on a nested competitive quantitative PCR the 16S rDNA of the commensal thermophile was only detected in compost soils at about $1.0{\times}10^4$ cpoies per gram of soil, corresponding to $0.25{\times}10^4$ cells per gram of soil. However, in an enrichment experiment at $60^{\circ}C$, about $1.0{\times}10^8$ copies of 16S rDNA molecules were detected per ml of enriched culture broth for all the soils, and more than 0.1 mM indole accumulated as the product of commensal bacterial growth. When incubated at $30^{\circ}C$, neither the 16S rDNA of the commensal bacterium nor any indole accumulation was detected. Accordingly, even though the 16S rDNA of the bacterium was only detected in the compost soils by a nested PCR, the presence of the 16S rDNA molecules of commensal thermophile and accumulation of indole in all the enriched cultures appeared to indicate that the commensal thermophile is widely distributed in various soils.

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Comparison of Histopathology, Serology and PCR for the Diagnosis of Malignant Catarrhal Fever (Malignant Catarrhal Fever의 병리조직학적 진단과 혈청학적 진단 및 PCR 진단법의 비교)

  • Kim, Ok-jin;Crawford, Timothy B.
    • Korean Journal of Veterinary Research
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    • v.43 no.3
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    • pp.471-476
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    • 2003

  • Malignant catarrhal fever (MCF) is a systemic disease of ruminants caused by ovine herpesvirus 2 (OvHV-2). OvHV-2 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens and even specific nucleic acids because of its low viral copies in the infected tissues. Histopathology, serology and polymerase chain reaction (PCR) were compared for the diagnosis of MCF using 10 bison infected with OvHV-2. Histopathological diagnosis was performed using the criteria which was based upon the pathognomic lesions. Serological diagnosis was conducted using its serum with competitive ELISA for the detection of antibodies of OvHV-2. Also, the nest PCR was performed with peripheral blood leukocytes for the detection of OvHV-2-specific DNAs. Primers 556 and 775 were used for the primary amplification, and primers 556 and 555 were used for the secondary amplification. As the results, positive cases were 6 by histopahology, 9 by serology and 10 by PCR. As comparing with other diagnostic methods, PCR was found to be more sensitive than histopathology and serology. The recent development of molecular diagnostic assays has provided powerful tools for investigating how viruses survive in nature. Development of PCR specific for viruses has dramatically improved the accuracy of diagnosis of viruses in clinically infected animals. Furthermore, amplification of viral genomic material by nest PCR represents the most sensitive method for the detection of viruses and might be detected successfully even though very low viral DNA copies. So, it could be used as the first choice for the detection of viral DNAs with low copies such as the status of latent infection. However, it has also some limitation of application like as false negative results by PCR inhibitors and false positive results by contamination. The results of this study suggest that the use of molecular biological methods like PCR may increase the accuracy for the diagnosis of infectious diseases. However, in diagnostic laboratory, it is recommended that PCR assay must be conducted with other diagnostic methods for more reliable diagnosis.

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