• Title/Summary/Keyword: RFLP.

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An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

  • Hawash, Yoursry;Ghonaim, M.M.;Al-Shehri, S.S.
    • Parasites, Hosts and Diseases
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    • v.54 no.1
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    • pp.1-8
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    • 2016
  • Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, $RIDA^{(R)}$ Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

Utilization of Porcine Clotted and Dried Blood for Estrogen Receptor Gene PCR-RFLP (에스트로겐 수용체 유전자의 다형 현상 추정을 위한 응고 및 건조된 돼지 혈액의 이용)

  • Seo, D.S.;Yang, S.H.;Park, H.B.;Park, S.S.;Hong, K.C.;Ko, Y.
    • Korean Journal of Animal Reproduction
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    • v.23 no.2
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    • pp.159-163
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    • 1999
  • Recent development of the molecular biology techniques has made it possible to characterize and analyze early diagnoses of genetic disorders and economic trait loci. In this study, porcine genomic DNA was extracted from both clotted and dried blood to analyze the porcine estrogen receptor (ER) gene by polymerase chain reaction (PCR). By the methods reported here, genomic DNA extracted from clotted or dried blood was efficient enough to detect ER gene by PCR. Moreover, the PCR-RFLP (restriction fragment length polymorphism) of ER gene was identified by PvuII restriction enzyme. Thus the results obtained from this study show that the clotted and dried blood was useful for identification of the certain genotype in a rapid manner with low cost. Importantly, this study implies that the whole blood can be economically utilized in studies of endocrinology, molecular biology, and genetics by obtaining both serum and DNA simultaneously in an efficient manner.

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Specific Marker Gene Analyses for DNA Polymorphism of the Blood Cell in Korea Native Brindled Cattle (칡한우 혈액에서 DNA 다양성 분석을 통한 표지 유전자 탐색)

  • Kim, Sang-Hwan;Hong, Yeon-Sik;Lee, Ho-Joun;Yoon, Jong-Taek
    • Development and Reproduction
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    • v.15 no.4
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    • pp.315-324
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    • 2011
  • This study was conducted to detect the specific expressing genes by using RAPD-PCR and RFLP method in the Korea Native Brindled Cattle, Korean Native cow and Holstein cattle. And then, the specific marker gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korea Native Brindled Cattle. It was detected the differences of the species in the colour and external section. The Korea Native Brindled Cattle were vary low compare to the Korean Native cow and Holstein cattle by analysis result of polymorphism and distribution. And there were a found the specific marker gene by sequencing in the R9B gene fragment of Korea Native Brindled Cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific marker gene in the Korea Native Brindled Cattle.

The PGC-II Polymorphism of the Peroxisome Proliferator-activated Receptor ${\gamma}$ Coactivator $1{\alpha}$ (PGC-$1{\alpha}$) Gene in Korean Subjects with the Metabolic Syndrome

  • Im, Sun-Ok;Kim, In-Sik;Kang, Sang-Sun;Hyun, Sung-Hee
    • Biomedical Science Letters
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    • v.18 no.2
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    • pp.139-145
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    • 2012
  • RFLP of PGC-$1{\alpha}$ gene of 285 Korean women was analyzed by PCR and HpaII restriction. We evaluated the correlation between PGC 1 genotypes and biochemical results, using the results of RFLP. Study subjects were divided into 3 groups: normal group (who has been average value of serum biochemical analysis), upper group (who has been higher value than average value), and low group (who have been lower value than average value). The frequencies of $H_1H_1$, $H_1H_2$, and $H_2H_2$ genotypes were 92 (32%), 85 (32%), and 108 (38%) respectively, and the ratio between $H_1$ and $H_2$ alleles was 1:1.1. There were no meaningful differences between biochemical results and PGC-$1{\alpha}$ genotypes in the normal group. But, in upper group, there was significant difference in total cholesterol (P=0.04) level. In the result of Turkey multiple comparison test, the P value of $H_1H_1$ and $H_2H_2$ was 0.059. In upper group, there were noticeable differences also in triglyceride (P=0.034) level and glucose (P=0.043) level, respectively. There were important differences between $H_1H_1$ type and $H_1H_2$ type in triglyceride (P=0.029) level and between $H_1H_2$ type and $H_2H_2$ type in glucose (P=0.040) level. This study may provide the PGC-$1{\alpha}$ genotype patterns for the amounts of lipid and glucose in the serum. $H_2$ allele (Ser482) of PGC-$1{\alpha}$ gene may be related with upper group in Korean women.

Investigation of Genetic Diversity of Fusarium oxysporum f. sp. fragariae Using PCR-RFLP

  • Kim, Ji-Su;Kang, Nam Jun;Kwak, Youn-Sig;Lee, Choungkeun
    • The Plant Pathology Journal
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    • v.33 no.2
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    • pp.140-147
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    • 2017
  • Fusarium wilts of strawberry, caused by Fusarium oxysporum f. sp. fragariae, is a serious soil-borne disease. Fusarium wilt causes dramatic yield losses in commercial strawberry production and it is a very stubborn disease to control. Reliable chemical control of strawberry Fusarium wilt disease is not yet available. Moreover, other well-known F. oxysporum have different genetic information from F. oxysporum f. sp. fragariae. This analysis investigates the genetic diversity of strawberry Fusairum wilt pathogen. In total, 110 pathogens were isolated from three major strawberry production regions, namely Sukok, Hadong, Sancheong in Gyeongnam province in South Korea. The isolates were confirmed using F. oxysporum f. sp. fragariae species-specific primer sets. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses were executed using the internal transcribed spacer, intergenic spacer, translation elongation factor1-${\alpha}$, and ${\beta}$-tubulin genes of the pathogens and four restriction enzymes: AluI, HhaI, HinP1I and HpyCH4V. Regarding results, there were diverse patterns in the three gene regions except for the ${\beta}$-tubulin gene region. Correlation analysis of strawberry cultivation region, cultivation method, variety, and phenotype of isolated pathogen, confirmed that genetic diversity depended on the classification of the cultivated region.

Molecular Biological Diagnosis of Meloidogyne Species Occurring in Korea

  • Oh, Hyung-Keun;Bae, Chang-Hwan;Kim, Man-Il;Wan, Xinlong;Oh, Seung-Han;Han, Yeon-Soo;Lee, Hyang-Burm;Kim, Ik-Soo
    • The Plant Pathology Journal
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    • v.25 no.3
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    • pp.247-255
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    • 2009
  • Root-knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria, and M. javanica are the most economically notorious nematode pests, causing serious damage to a variety of crops throughout the world. In this study, DNA sequence analyses were performed on the D3 expansion segment of the 28S gene in the ribosomal DNA in an effort to characterize genetic variations in the three Meloidogyne species obtained from Korea and four species from the United States. Further, PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) PCR and RAPD (Randomly Amplified Polymorphic DNA) were also utilized to develop methods for the accurate and rapid species identification of the root-knot nematode species. In the sequence analysis of the D3 expansion segment, only a few nucleotide sequence variations were detected among M. incognita, M. arenaria, and M, javanica, but not M. hapla. As a result of our haplotype analysis, haplotype 5 was shown to be common in M. arenaria, M. incognita, M. javanica, but not in the facultatively parthenogenetic species, M. hapla. PCR-RFLP analysis involving the amplification of the mitochondrial COII and large ribosomal RNA (lrRNA) regions yielded one distinct amplicon for M. hapla at 500 bp, thereby enabling us to distinguish M. hapla from M. incognita, M. arenaria, and M. javanica reproduced via obligate mitotic parthenogenesis. SCAR markers were used to successfully identify the four tested root-knot nematode species. Furthermore, newly attempted RAPD primers for some available root-knot nematodes also provided some species-specific amplification patterns that could also be used to distinguish among root-knot nematode species for quarantine purposes.

Enrichment of Ammonia-Oxidizing Bacteria for Efficient Nitrification of Wastewater

  • KIM WON-KYOUNG;CUI RONG;JAHNG DEOKJIN
    • Journal of Microbiology and Biotechnology
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    • v.15 no.4
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    • pp.772-779
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    • 2005
  • Ammonia-oxidizing bacteria (AOB) were enriched by repeating fed-batch cultivations in an AOB-selective medium of activated sludges from a domestic wastewater treatment plant. Enriched culture showed strong capabilities of ammonia oxidation [0.810 mg $NH_4^+$-N/mg mixed liquor suspended solids (MLSS)$\cdot$day] as well as $NO_x^-$-N production (0.617 mg $NO_x^-$-N/ mg MLSS$\cdot$day). Degree of enrichment was examined through fluorescent in situ hybridization (FISH) analyses using an AOB-specific Cy3-labeled oligonucleotide probe (NSOl90) and terminal-restriction fragment length polymorphism (T-RFLP) analyses. FISH analyses confirmed that the fraction of AOB among 4',6-diamidino-2-phenylindole (DAPI)-stained cells increased from about less than $0.001\%$ to approximately $42\%$ after enrichment of AOB, and T-RFLP analyses showed that bacterial community became simpler as enrichment was continued. When the enriched culture of AOB was added (150 mg/l as dry suspended solid) to the normal activated sludge (3,000 mg/l as dry suspended solid), nitrification efficiencies were improved from 0.020 mg $NO_x^-$-N/mg MLSS$\cdot$day to 0.041 mg $NO_x^-$-N/mg MLSS$\cdot$day in a synthetic wastewater and also from 0.0007 mg $NO_x^-$-N/mg MLSS$\cdot$day to 0.0918 mg $NO_x^-$-N/mg MLSS$\cdot$day in a real domestic wastewater. Therefore, it is expected that this enrichment method could be used for improving efficiency of nitrification in wastewater treatment plants.

Evaluation of Lactic Acid Bacterial Community in Kimchi Using Terminal-Restriction Fragment Length Polymorphism Analysis (Terminal-Restriction Fragment Length Polymorphism 분석을 이용한 김치발효 관련 유산균 군집의 평가)

  • Shim, Sang-Min;Lee, Jong-Hoon
    • Microbiology and Biotechnology Letters
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    • v.36 no.4
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    • pp.247-259
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    • 2008
  • Terminal-restriction fragment length polymorphism (T-RFLP) analysis, one of rapid culture-independent microbial community analysis methods, was used to determine the lactic acid bacterial complexity and dynamics during kimchi fermentation at $15^{\circ}C$ and $4^{\circ}C$. At both temperatures, the common presence of Leuconostoc mesenteroides, Lc. inhae, Lc. kimchi, Weissella koreensis, W. cibaria, Lactobacillus sakei, Lb. curvatus, Lb. plantarum, Lb. paraplantarum, Lb. pentosus, and Lb. brevis was predicted. Lc. citreum and Enterococcus faecalis were detected at $15^{\circ}C$ and $4^{\circ}C$, respectively. W. koreensis predominated during the mid stage of kimchi fermentation whereas lactobacilli were dominants during later stage. Lb. sakei and Lb. curvatus became dominants regardless of fermentation temperature but the growth of Lb. plantarum, Lb. paraplantarum, Lb. pentosus, and Lb. brevis was restricted at psychrophilic temperature. Some species of leuconostocs were maintained until the later stage of kimchi fermentation.