• Title/Summary/Keyword: AS-PCR

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Convenient Genetic Diagnosis of Virion Captured (VC)/RT-PCR for Rice Viruses (RSV, RBSDV) and Small Brown Plant Hopper (벼 바이러스(RSV, RBSDV)와 애멸구의 간편한 VC/RT-PCR 유전자 진단기술)

  • Kim, Jeong-Soo;Lee, Su-Heon;Choi, Hong-Soo;Cho, Jeom-Deog;Noh, Tai-Whan;Kim, Jin-Young
    • Research in Plant Disease
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    • v.15 no.2
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    • pp.57-62
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    • 2009
  • Genetic diagnosis method of Virion Captured (VC)/RT-PCR for Rice stripe virus (RSV) and Rice black-streaked dwarf virus (RBSDV), Korean major rice viruses transmitted by small brown plant hopper, Laodelphax striatellus, was developed. Virion extraction buffer for rice plant was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. However, the extraction buffer for L. striatellus was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite and 2% polyvinylpyrrolidone wt 40,000 (PVP-40). Specific primers for detection of RSV and RBSDV were selected for VC/RT-PCR method. The specific primers were used as a duplex primer to detect viruliferous small brown plant hopper collected from Gimpo, Pyeongtaek and Siheung areas in Gyeonggi province. The genetic diagnosis methods of single and duplex VC/RT-PCR for RSV and RBSDV could be used easily and economically, especially on the diagnosis of L. striatellus. The rate of viruliferous insect (RVI) for RSV was compared with ELISA and VC/RT-PCR for L. striatellus collected from fields. RVI by ELISA was same as 9.2% with RVI by VC/RT-PCR. However, there were some different detection results between the methods. It could be suggested that there is a possibility of serological and/or genomic differences among RSV isolates. The portion of RVI detected simultaneously by ELISA and VC/RT-PCR was 71.0%, and the detection rate from VC/RT-PCR was higher as 3.2% than that from ELISA, which had a reason of simultaneous detection ability both RSV and RBSDV of VC/RT-PCR.

Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR for the Rapid Detection of Salmonella Typhimurium, Listeria monocytogenes and Cronobacter sakazakii Artificially Inoculated in Foods (식품에 인위접종된 Salmonella Typhimurium, Listeria monocytogenes, Cronobacter sakazakii의 신속검출을 위한 Real-time PCR과 Loop-mediated isothermal amplification 비교)

  • Kim, Jin-Hee;Oh, Se-Wook
    • Journal of Food Hygiene and Safety
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    • v.34 no.2
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    • pp.135-139
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    • 2019
  • The objective of this research was to compare loop-mediated isothermal amplification (LAMP) with real-time polymerase chain reaction (PCR) for the rapid detection of pathogens in foods. In this study, the limits of detection (LODs) for Salmonella Typhimurium, Listeria monocytogenes, and Cronobacter sakazakii were evaluated in various foods. Among 11 samples tested for S. Typhimurium, LAMP and real-time PCR had the same LODs in beef and duck meat whereas real-time PCR was more sensitive than the LAMP in 8 samples. However, S. Typhimurium in chocolate samples was not detected by real-time PCR. The sensitivity of real-time PCR was high in all samples inoculated with L. monocytogenes and C. sakazakii whereas LAMP was more sensitive than real-time PCR in oil-rich foods. Therefore, LAMP can be shown as an easrer, more convenient method, as well as effective analytical method for testing difficult samples when employed in PCR.

Improvement of polymerase chain reaction methods for rapid detection of Listeria monocytogenes in raw milk (원유로부터 Listeria monocytogenes의 신속검색을 위한 종합효소 연쇄반응법의 개선)

  • Yi, Chul-hyun;Son, Won-geun;Kang, Ho-jo
    • Korean Journal of Veterinary Research
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    • v.36 no.1
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    • pp.119-129
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    • 1996
  • The present study was conducted to rapidly detect Listeria monocytogenes in raw milk. Specificity and sensitivity of polymerase chain reaction(PCR) technique, and direct PCR were examinded in raw milk, also were compared the calssical culture methods with PCR technique. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L nomocytogenes. In the PCR specificity tests, each of the 10 strains of L monocytogenes tested gave a single 70-bp band. But the other six Listera spp tested gave negative results. Results of the sensitivity tests showed that as few as 2 CFU of L monocytogenes in pure cultures could be detected with 16S rRNA-based primers, L-1 and L-2. In different PCR cycles, a PCR product was detected with $10^3$ cells of L monocytogenes from 25 cycles to 50 cycles and the concentration of PCR products was cycle-dependent. Raw milk samopes added L monocytogenes cells gave negative results. However, these samplers gave a single 70-bp band by pretreatment of pronase, and PCR products were detected with $10^1$ cells of L monocytogenes. To detemine the most sensitive culture protocol to use in conjunction with the PCR assay, raw milk samples were inoculated with L monocytogenes at concentrations ranging from 1 to $5.7{\times}10^4CFU/ml$. PCR assays from Listeria enrichment broth(LEB) containing raw milk samples added L monocytogene EGD could dtect 10 cells in pronase-pretreated samples without incubation, and 1 cell of L monocytogenes in both 12 hr and 24 hr incubation, respectively. Isolation raw of PCR assays was similar to that of classical culture methods, but required time for detection of L monocytogenes could remarkably be reduced compare to culture methods.

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The Effect of Temperature and Cycles on Amplification of DNA by PCR (PCR에 의한 DNA 증폭에 미치는 온도와 Cycle 수)

  • Kim, Chong-Ho;Shin, Sang-Hee
    • Korean Journal of Clinical Laboratory Science
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    • v.36 no.1
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    • pp.33-37
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    • 2004
  • In order to study the effect of temperature of denaturation, annealing and extension and cycles on amplification of DNA by PCR method, We isolated the hepatitis B virus DNA from hepatitis B patient blood and compared the density of DNA amplified by Reference PCR Program (denaturation at $94^{\circ}C$ for 30 sec., annealing at $60^{\circ}C$ for 1 min., extension at $72^{\circ}C$ for 1 min., holding at $72^{\circ}C$ for 5min., 30 cycles) that is usually used in laboratory to the density of DNA amplified by PCR program changed only the denaturation temperature or annealing temperature or extension temperature. We amplified about 341bp of hepatitis B virus DNA by Reference PCR Program from hepatitis patient blood, but the DNAs denatured at $72^{\circ}C$ or $60^{\circ}C$ were not detectable on photoradiography film. The DNA amplified at $37^{\circ}C$ of annealing temperature was not detectable, but the DNA annealed at $72^{\circ}C$ was detectable the lower density of DNA than the DNA amplified by Reference PCR Program. Each DNA amplified by PCR program changed only the extension temperature to $37^{\circ}C$ or $60^{\circ}C$ was almost same density as DNA amplified by Reference PCR Program. We compared the density of hepatitis B virus DNA amplified by Reference PCR Program for 30 cycles, 20 cycles, 10 cycles, and 5 cycles. The DNA cycled for 20 cycles was not amplified well as cycled for 30 cycles, but the DNA was detectable on the photoradiography film. The DNAs amplified for 10 cycles or 5 cycles were not detectable on photoradiorgaphy film. The concentration of hepatitis B virus DNA amplified in Reference PCR condition for 30 cycles, 20 cycles, 10 cycles, and 5 cycles were $72{\mu}g/m{\ell}$, $83{\times}10^{-3}{\mu}g/m{\ell}$, $27{\times}10^{-6}{\mu}g/m{\ell}$, and nondetectable, respectively.

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Characterization of beer-spoilage microorganism and its rapid detection by specific PCR primer (맥주오염미생물의 동정과 specific PCR primer의한 신속한 검출 방법)

  • Lee, Taek-In;Choi, Shin-Geon
    • Journal of Industrial Technology
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    • v.28 no.A
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    • pp.141-147
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    • 2008
  • Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

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Optimization of DNA Extraction and PCR Conditions for Fungal Metagenome Analysis of Atmospheric Particulate Matter (대기 입자상물질 시료의 곰팡이 메타게놈 분석을 위한 DNA 추출 및 PCR 조건 최적화)

  • Sookyung Kang;Kyung-Suk Cho
    • Microbiology and Biotechnology Letters
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    • v.51 no.1
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    • pp.99-108
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    • 2023
  • Several challenges arise in DNA extraction and gene amplification for airborne fungal metagenome analysis from a particulate matter (PM) samples. In this study, various conditions were tested to optimize the DNA extraction method from PM samples and polymerase chain reaction (PCR) conditions with primer set and annealing temperature. As a result of comparative evaluation of DNA extraction under various conditions, chemical cell lysis using buffer and proteinase K for 20 minutes and bead beating treatment were followed by using a commercial DNA extraction kit to efficiently extract DNA from the PM filter samples. To optimize the PCR conditions, PCR was performed using 10 primer sets for amplifying the ITS2 gene region. The concentration of the PCR amplicon was relatively high when the annealing temperature was 58℃ with the ITS3tagmix3/ITS4 primer set. Even under these conditions, when the concentration of the PCR product was low, nested PCR was performed using the primary PCR amplicon as the template DNA to amplify the ITS2 gene at a satisfactory concentration. Using the methods optimized in this study, DNA extraction and PCR were performed on 15 filter samples that collected PM2.5 in Seoul, and the ITS2 gene was successfully amplified in all samples. The optimized methods can be used for research on analyzing and interpreting the fungal metagenome of atmospheric PM samples.

Development of real-time PCR for rapid detection of Mycobacterium bovis DNA in cattle lymph nodes and differentiation of M. bovis and M. tuberculosis (소 림프절에서 Mycobacterium bovis DNA의 신속 검출과 M. bovis와 M. tuberculosis 감별을 위한 real-time PCR 개발)

  • Koh, Ba-Ra-Da;Jang, Young-Boo;Ku, Bok-Kyung;Cho, Ho-Seong;Bae, Seong-Yeol;Na, Ho-Myung;Park, Seong-Do;Kim, Yong-Hwan;Mun, Yong-Un
    • Korean Journal of Veterinary Service
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    • v.34 no.4
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    • pp.321-331
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    • 2011
  • Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

A Simple Detection of Sweetpotato Feathery Mottle Virus by Reverse Transcription Polymerase Chain Reaction

  • Jeong Jae-Hun;Chakrabarty Debasis;Kim Young-Seon;Eun Jong-Seon;Choi Yong-Eui;Paek Kee-Yoeup
    • Journal of Plant Biotechnology
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    • v.5 no.2
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    • pp.83-86
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    • 2003
  • A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

Direct Detection of Cylindrocarpon destructans, Root Rot Pathogen of Ginseng by Nested PCR from Soil Samples

  • Jang, Chang-Soon;Lim, Jin-Ha;Seo, Mun-Won;Song, Jeong-Young;Kim, Hong-Gi
    • Mycobiology
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    • v.38 no.1
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    • pp.33-38
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    • 2010
  • We have successfully applied the nested PCR to detect Cylindrocarpon destructans, a major pathogen causing root rot disease from ginseng seedlings in our former study. The PCR assay, in this study, was used to detect the pathogen from soils. The nested PCR using internal transcribed spacer (ITS) 1, 4 primer set and Dest 1, 4 primer set maintained the specificity in soils containing various microorganisms. For a soil DNA extraction method targeting chlamydospores, when several cell wall disrupting methods were tested, the combination of lyophilization and grinding with glass beads, which broke almost all the chlamydospores, was the strongest. The DNA extraction method which was completed based on the above was simple and time-saving because of exclusion of unnecessary stages, and efficient to apply in soils. As three ginseng fields whose histories were known were analyzed, the PCR assay resulted as our expectation derived from the field information. The direct PCR method will be utilized as a reliable and rapid tool for detecting and monitoring C. destructans in ginseng fields.

Molecular Detection and Analysis of Sweet potato feathery motile vims from Root and Leaf Tissues of Cultivated Sweet Potato Plants

  • Ryu, Ki-Hyun;Park, Sun-Hee
    • The Plant Pathology Journal
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    • v.18 no.1
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    • pp.12-17
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    • 2002
  • For the molecular detection of Sweet potaio feathery mottle virus (SPFMV) from diseased sweet potato plants, reverse transcription and polymerase chain reaction (RT-PCR) was performed with the use of a set of virus-specific primers to amplify an 816 bp product. The viral coat protein gene was selected for the design of the primers. No PCR product was amplified when Turnip mosaic virus, Potato vims Y or Cucumber mosaic virus were used as template in RT-PCR with the SPFMV-specific primers. The lowest concentration of template viral RNA required for detection was 10 fg. The vim was rapidly detected from total nucleic acids of leaves and roots from the virus-infected sweet potato plants as well as from the purified viral RNA by the RT-PCR. Twenty-four sweet potato samples were selected and analyzed by RT-PCR and restriction fragment length polymorphism (RFLP). RFLP analysis of the PCR products showed three restriction patterns, which resulted in some point mutations suggesting the existence of quasi-species for the vims in the infected sweet potato plants.