• Title/Summary/Keyword: ITS-PCR

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Molecular Identification of Vaginal Lactobacillus spp. Isolated from Korean Women

  • CHANG, CHUNG EUN;SYLVIA I. PAVLOVA;LIN TAO;EUN-KI KIM;SEUNG CHUL KIM;HYUN SHIK YUN;JAE-SEONG SO
    • Journal of Microbiology and Biotechnology
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    • v.12 no.2
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    • pp.312-317
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    • 2002
  • Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.

Simultaneous Detection of Food-borne Pathogenic Bacteria in Ready-to-eat Kimbab Using Multiplex PCR Method

  • Cho, Kye-Man;Kambiranda, Devaiah M;Kim, Seong-Weon;Math, Renukaradhya K;Lim, Woo-Jin;Hong, Su-Young;Yun, Han-Dae
    • Food Science and Biotechnology
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    • v.17 no.6
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    • pp.1240-1245
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    • 2008
  • Kimbab is the most popular ready-to-eat (RTE) food in Korea. A rapid detection method based on multiplex PCR technique was developed for detection of major food-borne pathogens like Salmonella spp., Shigella spp., Bacillus cereus, Listeria monocytongenes, and Staphylococcus aureus. Specific bands were obtained as 108 bp (Sau, S. aureus), 284 bp (Sal, S. enterica, S. enteritids, and S. typhmurium), 404 bp (Lmo, L. monocytogenes), 475 bp (Bce, B. cereus), and 600 bp (Shi, S. flexineri and S. sonnei). Visible cell numbers varied from 4.14-5.03, 3.61-4.47, and 4.10-5.11 log CFU/g in randomly collected June, July, and August samples, respectively. Among the 30 kimbab samples obtained 83.3% samples were contaminated and 16.7% samples were free from contamination. The highest rate of contamination was with S. aureus (56.7%) followed by B. cereus (43.3%), Salmonella spp. (36.7%), Shigella spp. (13.3%), and L. monocytogenes (6.7%). The identification of the pathogenic species could be faster using one polymerase chain reaction (PCR) and the ability to test for food-borne pathogenic species in kimbab will save time and increase the ability to assure its quality.

Performance Evaluation of the Automated Fluorescent Immunoassay System Rotavirus Assay in Clinical Samples

  • Kim, Jae-Seok;Lee, Su-Kyung;Ko, Dae-Hyun;Hyun, Jungwon;Kim, Hyun Soo
    • Annals of Laboratory Medicine
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    • v.39 no.1
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    • pp.50-57
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    • 2019
  • Background: The Automated Fluorescent Immunoassay System (AFIAS) rotavirus assay (Boditech Med Inc., Chuncheon, Korea) is a new rapid antigen test for rotavirus detection. We evaluated the performance of this assay for detecting rotaviruses and their specific genotypes in clinical stool samples. Methods: AFIAS rotavirus assay was performed in 103 rotavirus-positive and 103 rotavirus-negative stool samples (confirmed by both PCR and ELISA), and its results were compared with those of PCR, ELISA, and immunochromatographic assay (ICA). We evaluated diagnostic sensitivity/specificity, the detectability of rotavirus subtypes, lower limit of detection (LLOD), reproducibility, cross-reactivity, and interference of AFIAS rotavirus assay. Results: Based on PCR and ELISA results, diagnostic sensitivity and specificity of the AFIAS rotavirus assay were both 99.0%. LLOD results showed that the AFIAS assay had sensitivity similar to or greater than ICA and ELISA. High reproducibility was confirmed, and no cross-reactivity or interference was detected. This assay could detect genotypes G1P[8], G2P[4], G3P[8], G4P[6], G4P[8], G8P[4], G8P[8], G9P[4], and G9P[8]. Conclusions: The AFIAS rotavirus assay showed high reproducibility, sensitivity, and specificity as well as excellent agreement with ELISA, PCR, and ICA. It detected the most common as well as unusual genotypes of rotavirus prevalent in Korea. It could be a useful onsite assay for rapid, convenient, and cost-effective detection of rotavirus infection.

Prevalence of Bordetella bronchiseptica, Mycoplasma felis, and Chlamydia felis using a newly developed triplex real-time polymerase chain reaction assay in Korean cat population

  • Hye-Ryung, Kim;Gyu-Tae, Jeon;Jong-Min, Kim;Ji-Su, Baek;Yeun-Kyung, Shin;Oh-Kyu, Kwon;Hae-Eun, Kang;Ho-Seong, Cho;Doo-Sung, Cheon;Choi-Kyu, Park
    • Korean Journal of Veterinary Service
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    • v.45 no.4
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    • pp.305-316
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    • 2022
  • Bordetella (B.) bronchiseptica, Mycoplasma (M.) felis, and Chlamydia (C.) felis are considered as main bacterial pathogens of feline upper respiratory tract disease (URTD). In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) assay was developed for the rapid and differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with the detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1%. Based on the diagnostic results of the assay using 94 clinical samples obtained from cats with URTD signs, prevalence of B. bronchiseptica, M. felis, or C. felis was 10.6%, 36.2%, or 6.4%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported monoplex qPCR assays. The dual infection rates for B. bronchiseptica and M. felis or M. felis and C. felis was 2.1% or 3.2%, respectively. These results indicated that M. felis has been widely spread, and its co-infection with B. bronchiseptica or M. felis has been frequently occurred in Korean cat population. The developed tqPCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens and the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of feline URTD in Korea.

Unified Non-iterative Algorithm for Principal Component Regression, Partial Least Squares and Ordinary Least Squares

  • Kim, Jong-Duk
    • Journal of the Korean Data and Information Science Society
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    • v.14 no.2
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    • pp.355-366
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    • 2003
  • A unified procedure for principal component regression (PCR), partial least squares (PLS) and ordinary least squares (OLS) is proposed. The process gives solutions for PCR, PLS and OLS in a unified and non-iterative way. This enables us to see the interrelationships among the three regression coefficient vectors, and it is seen that the so-called E-matrix in the solution expression plays the key role in differentiating the methods. In addition to setting out the procedure, the paper also supplies a robust numerical algorithm for its implementation, which is used to show how the procedure performs on a real world data set.

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Development of Virtual Metrology Models in Semiconductor Manufacturing Using Genetic Algorithm and Kernel Partial Least Squares Regression (유전알고리즘과 커널 부분최소제곱회귀를 이용한 반도체 공정의 가상계측 모델 개발)

  • Kim, Bo-Keon;Yum, Bong-Jin
    • IE interfaces
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    • v.23 no.3
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    • pp.229-238
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    • 2010
  • Virtual metrology (VM), a critical component of semiconductor manufacturing, is an efficient way of assessing the quality of wafers not actually measured. This is done based on a model between equipment sensor data (obtained for all wafers) and the quality characteristics of wafers actually measured. This paper considers principal component regression (PCR), partial least squares regression (PLSR), kernel PCR (KPCR), and kernel PLSR (KPLSR) as VM models. For each regression model, two cases are considered. One utilizes all explanatory variables in developing a model, and the other selects significant variables using the genetic algorithm (GA). The prediction performances of 8 regression models are compared for the short- and long-term etch process data. It is found among others that the GA-KPLSR model performs best for both types of data. Especially, its prediction ability is within the requirement for the short-term data implying that it can be used to implement VM for real etch processes.

Inter Simple Sequence Repeat (ISSR) Polymorphism and Its Application in Mulberry Genome Analysis

  • Vijayan Kunjupillai
    • International Journal of Industrial Entomology and Biomaterials
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    • v.10 no.2
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    • pp.79-86
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    • 2005
  • Molecular markers have increasingly been used in plant genetic analysis, due to their obvious advantages over conventional phenotypic markers, as they are highly polymorphic, more in number, stable across different developmental stages, neutral to selection and least influenced by environmental factors. Among the PCR based marker techniques, ISSR is one of the simplest and widely used techniques, which involves amplification of DNA segment present at an amplifiable distance in between two identical microsatellite repeat regions oriented in opposite direction. Though ISSR markers are dominant like RAPD, they are more stable and reproducible. Because of these properties ISSR markers have recently been found using extensively for finger printing, pohylogenetic analysis, population structure analysis, varietal/line identification, genetic mapping, marker-assisted selection, etc. In mulberry (Morus spp.), ISSR markers were used for analyzing phylogenetic relationship among cultivated varieties, between tropical and temperate mulberry, for solving the vexed problem of identifying taxonomic positions of genotypes, for identifying markers associated with leaf yield attributing characters. As ISSR markers are one of the cheapest and easiest marker systems with high efficiency in generating polymorphism among closely related varieties, they would play a major role in mulberry genome analysis in the future.

Oligonucleotide Array-based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma)

  • Jang, Hyun-Jung;Kim, Hyo-Myeung;Kang, Byeong-Chul;Kim, Cheol-Min;Park, Hee-Kyung
    • Journal of Microbiology and Biotechnology
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    • v.19 no.3
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    • pp.265-270
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    • 2009
  • An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCR-RFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.

Sex Determination of Cattle Meat by Polymerase Chain Reaction Amplification of the DEAD Box Protein (DDX3X/DDX3Y) Gene

  • Gokulakrishnan, P.;Kumar, R.R.;Sharma, B.D.;Mendiratta, S.K.;Sharma, D.
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.5
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    • pp.733-737
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    • 2012
  • Determination of sex origin of cattle meat by fast and reliable molecular methods is an important measure to ensure correct allocation of export refunds particularly in European countries and also female cattle (cow) slaughter is legally banned in India because of religious beliefs. Based on the DEAD box protein gene located on the X and Y chromosomes, 2 pair of primers were designed and the system of PCR was optimized. Upon PCR amplification, male tissue showed 2 bands, while female tissue resulted in only one band. The accuracy and specificity of the primers was assessed using DNA template extracted from cattle meat of known sex. The protocol was subjected to a blind test and showed 100% concordance, proving its accuracy and reliability.

Cloning and expression of cellulase genes from Erwinia carotovora in E. coli (Erwinia carotovora 유래의 cellulase 유전자의 클로닝 및 대장균에서의 발현)

  • Kim, Se-Don;Choi, Shin-Geon
    • Journal of Industrial Technology
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    • v.29 no.B
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    • pp.121-125
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    • 2009
  • New cellulase genes, named as CelV2 and CelN1, respectively, were isolated from Erwinia carotovora ATCC15713 and expressed in E. coli. The CelV2 and CelN1 gene were PCR amplified with degenerated primers and PCR products were sequenced and expressed in E. coli. Two new cellulase genes showed 97% homologies with previously reported Erwinia cellulase genes. The recombinant cellulase were purified with Ni-NTA column chromatography and its enzymatic properties were characterized. The optimum temperature of two enzymes were about $50^{\circ}C$ degree and optimum pH were around pH7.0. The newly isolated celluase genes could be used for enhancing substrate range of alcohol-producing bacteria such as Zymomonas mobilis.

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