• Title/Summary/Keyword: G-S PCR

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Effects of Denaturants on the Conditions of Polymerase Chain Reactions with G+C-rich Primers (G+C 함량이 높은 Primer를 사용하는 중합효소 연쇄반응에서 변성제가 미치는 영향)

  • 김종배;안준환;엄용빈;김영미
    • Biomedical Science Letters
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    • v.2 no.2
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    • pp.241-247
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    • 1996
  • Poor yields of amplified DNAs could be resulted in polymerase chain reaction(PCR) processes with G+C-rich DNA primers because of their high $T_m$ values. To maximize the yields of amplification in PCR processes with G+C-rich primers, we compared the yields of amplified DNA fragments according to the concentrations of specific denaturants added to the reaction mixture of PCR system. With addition of the mixture of 2.5% glycerol and 1.25% formamide, or 2.5% dimethyl sulfoxide to the reaction cocktail, respectively, remarkable increases in the yields of amplified DNA fragments were not observed in the PCR systems with G+C-low primers of Lyl chromosomal gene from Borrelia burgdorferi but observed in the PCR system with G+C- ich primers of Is900 gene from Mycobacterium parahberculosis. Although we were not practically able to discriminate the yields of PCR DNAs according to the concentrations used in this study, addition of the mixture of 5% glycerol and 2.5% formamide, or 5% DMSO tended to increase the production of extra bands.

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Abundance and expression of denitrifying genes (narG, nirS, norB, and nosZ) in sediments of wastewater stabilizing constructed wetlands

  • Chon, Kyongmi;Cho, Jaeweon
    • Environmental Engineering Research
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    • v.20 no.1
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    • pp.51-57
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    • 2015
  • As expected, the expression of denitrifying genes in a Typha wetland (relatively stagnant compared to other ponds), showing higher nitrogen removal efficiency in summer, was affected by temperature. The abundance and gene transcripts of nitrate reductase (narG), nitrite reductase (nirS), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ) genes in seasonal sediment samples taken from the Acorus and Typha ponds of free surface flow constructed wetlands were investigated using quantitative polymerase chain reaction (Q-PCR) and quantitative reverse transcription PCR (Q-RT-PCR). Denitrifying gene copy numbers ($10^5-10^8$ genes $g^{-1}$ sediment) were found to be higher than transcript numbers-($10^3-10^7$ transcripts $g^{-1}$ sediment) of the Acorus and Typha ponds, in both seasons. Transcript numbers of the four functional genes were significantly higher for Typha sediments, in the warm than in the cold season, potentially indicating greater bacterial activity, during the relatively warm season than the cold season. In contrast, copy numbers and expression of denitrifying genes of Acorus did not provide a strong correlation between the different seasons.

PCR Detection and Sequence Analysis of the rDNA ITS Regions of Rhizina undulata (Rhizina undulata rDNA ITS 영역의 PCR 검정 및 염기배열 분석)

  • Lee, Sun Keun;Lee, Jong Kyu;Kim, Kyung Hee;Lee, Seung Kyu;Lee, Sang Yong
    • Journal of Korean Society of Forest Science
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    • v.96 no.4
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    • pp.425-431
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    • 2007
  • To investigate genetic diversity and PCR detection of Rhizina undulata, PCR detection and sequence analysis of rDNA ITS region of R. undulata in soil were analyzed and developed. The length of partial 18S rDNA from four R. undulata isolates were 1,375 nt. The sequence similarity of R. undulata isolates was 100%. The rDNA ITS regions of R. undulata isolates were 585 nt long. Nucleotide sequencing of the ITS regions showed that PDK-1, PTT-1 and PDJ-9 isolates had 100% sequence identity. But, PDS-5 isolate differed from the three isolates by two nucleotide substitution. R. undulata-specific primers designed by the sequence of ITS region were used in PCR detection of R. undulata. PCR products about 525 bp size, which is specific to R. undulata, were amplified from total DNAs of R. undulata isolates. To assay the sensitivity of PCR detection by R. undulata ITS-specific primer, purely cultured mycelial suspension of R. undulata was serially diluted and mixed with 100g of sterile sandy loam soil, respectively. And then, PCR products of total DNAs extracted from each mycelium-soil mixtures were analysed. The PCR protocol could detected up to 1ng mycelium of R. undulata within 100g of soil.

Rapid Detection of Escherichia coli in Fresh Foods Using a Combination of Enrichment and PCR Analysis

  • Choi, Yukyung;Lee, Sujung;Lee, Heeyoung;Lee, Soomin;Kim, Sejeong;Lee, Jeeyeon;Ha, Jimyeong;Oh, Hyemin;Lee, Yewon;Kim, Yujin;Yoon, Yohan
    • Food Science of Animal Resources
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    • v.38 no.4
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    • pp.829-834
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    • 2018
  • The objective of this study was to determine the minimum enrichment time for different types of food matrix (pork, beef, and fresh-cut lettuce) in an effort to improve Escherichia coli detection efficiency. Fresh pork (20 g), beef (20 g), and fresh-cut lettuce (20 g) were inoculated at 1, 2, and 3 Log CFU/g of Escherichia coli. Samples were enriched in filter bags for 3 or 5 h at $44.5^{\circ}C$, depending on sample type. E. coli cell counts in the samples were enriched in E. coli (EC) broth at 3 or 5 h. One milliliter of the enriched culture medium was used for DNA extraction, and PCR assays were performed using primers specific for uidA gene. To detect E. coli (uidA) in the samples, a 3-4 Log CFU/mL cell concentration was required. However, E. coli was detected at 1 Log CFU/g in fresh pork, beef, and fresh-cut lettuce after 5, 5, and 3-h enrichment, respectively. In conclusion, 5-h enrichment for fresh meats and 3-h enrichment for fresh-cut lettuce in EC broth at $44.5^{\circ}C$, and PCR analysis using uidA gene-specific primers were appropriate to detect E. coli rapidly in food samples.

Detection of 23S rRNA Mutation Associated with Clarithromycin Resistance in Children with Helicobacter pylori Infection (소아 Helicobacter pylori 감염에서 Clarithromycin 내성과 연관된 23S rRNA의 돌연변이)

  • Ko, Jae Sung;Yang, Hye Ran;Seo, Jeong Kee
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.7 no.2
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    • pp.137-142
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    • 2004
  • Purpose: The resistance of H. pylori to clarithromycin is one of the major causes of eradication failure. In H. pylori, clarithromycin resistance is due to point mutation in 23S rRNA. The aims of this study were to investigate the mutation of 23S rRNA and to examine the association of cagA, vacA genotype and clarithromycin resistant genes. Methods: H. pylori DNA was extracted from antral biopsy specimens from 27 children with H. pylori infection. Specific polymerase chain reaction (PCR) assays were used for cagA and vacA. Mutations associated with clarithromycin resistance were detected by using PCR restriction fragment length polymorphism (RFLP) analysis of 23S rRNA gene. Results: A2143G mutation was detected in one case and A2144G in 4, indicating 18.5% were clarithromycin resistant. Among the total of 27, cagA was present in 25 (93%), vacA s1a/m1 in 6 (22%), s1a/m2 in 3 (11%), s1c/m1 in 16 (59%), and s1c/m2 in 1 (4%). All of the 5 clarithromycin resistant strains were cagA (+), among which 2 were s1a/m1 and 2 were s1c/m1. There was no relation between genotypes and clarithromycin resistant genes. Conclusion: Detection of H. pylori resistance to clarithromycin using PCR RFLP from biopsy specimens might be useful for the selection of antibiotics. Clarithromycin resistant genes are not associated with genotypes of cagA and vacA.

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Specific Detection of Serratia marcescens Based on a PCR Assay and Antimicrobial Susceptibility of S. marcescens Isolated from Boar Semen (Serratia marcescens 검출을 위한 PCR 기법 개발 및 돼지정액 유래균주에 대한 항생제 감수성 양상)

  • Jung, Ji-A;Kim, Aeran;Seo, Byoung Joo;Jung, Suk Chan;Kim, In Cheul;Chung, Ki Hwa;Jung, Byeong Yeal
    • Journal of Life Science
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    • v.23 no.9
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    • pp.1133-1139
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    • 2013
  • During the collection of boar semen, bacterial contamination usually occurs. The contamination has deleterious effects both on semen quality and on sow fertility. The majority of contaminants are gram-negative bacteria, especially Serratia marcescens. In this study, we developed a PCR assay for the identification of S. marcescens targeting the luxS gene (GenBank no. EF164926). S. marcescens yielded a specific 306 bp PCR product. However, no amplification was observed in the other strains tested. The detection limit of PCR was $50pg/{\mu}l$ of template DNA of S. marcescens. The antimicrobial susceptibility patterns of S. marcescens isolated from boar semen were tested using the disk diffusion method. Gentamicin, ceftiofur, florfenicol, and neomycin showed high sensitivity in this test. The minimum inhibitory concentration (MIC) was also determined by the broth microdilution method. The $MIC_{90}$ values of ceftiofur, enrofloxacin, gentamicin, and neomycin were 8, 8, 8, and $16{\mu}g/ml$, respectively. These results indicate that PCR amplification of the luxS gene is a reliable and effective method for the identification of S. marcescens and that ceftiofur, enrofloxacin, gentamicin, and neomycin are effective semen extenders for controlling S. marcescens.

Detection of Vibrio vulnificus by Real-Time PCR targeted to rpoS gene (rpoS 유전자를 대상으로 하는 Real-Time PCR에 의한 Vibrio vulnificus 검출)

  • Kim, Dong-Gyun;Ahn, Sun-Hee;Bae, Ju-Yoon;Kong, In-Soo
    • Journal of Marine Bioscience and Biotechnology
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    • v.2 no.4
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    • pp.263-266
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    • 2007
  • Vibrio vulnificusis a causative agent of serious diseases in humans resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium using the rpoS gene in pure cultures and in infected oyster tissues.

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Comparison of Loop-mediated Isothermal Amplification and Korea Standard Food Codex (KFSC) Method for Detection of Salmonella Typhimurium, Listeria monocytogenes Artificially Inoculated in Yuk-hwe and Yuk-sashimi (육회와 육사시미에 접종된 Salmonella Typhimurium와 Listeria monocytogenes 검출을 위한 Loop-mediated isothermal amplification와 식품공전의 배지 시험법, real-time PCR의 검출 성능 비교)

  • Gwak, Seung-Hae;Lee, So-Young;Kim, Jin-Hee;Oh, Se-Wook
    • Journal of Food Hygiene and Safety
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    • v.34 no.3
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    • pp.277-282
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    • 2019
  • The object of this study is to compare the performance of the 3M Molecular Detection Assay 2 (3M MDA 2) and the Korea Standard Food Codex (KSFC) Method (i.e., isolation media and real-time PCR) in detecting Salmonella Typhimurium and Listeria monocytogenes in traditional Korean foods. Yuk-hwe and Yuk-sashimi (types of raw beef dishes) were artificially inoculated with $10^0-10^4CFU/25g$ of L. monocytogenes and S. Typhimurium. Citrobacter freundii and Listeria innocua were used as competitive microflora. After enrichment, the samples were analyzed using 3M MDA 2 and real-time PCR. All samples inoculated at concentrations of $10^0-10^4CFU/25g$ without competitive microflora were positive for S. Typhimurium and L. monocytogenes, as detected by 3M MDA 2 and Korea Standard Food Codex (KFSC) Method. In addition, part of the samples were positive for the presence of C. freundii and L. innocua. The 3M MDA 2 - Salmonella and Korea Standard Food Codex (KFSC) Method showed similar detection performances in Yuk-hwe and Yuk-sashimi. The 3M MDA 2 method for Salmonella and Listeria, which is a LAMP-based technology, can be used for rapid detection of S. Typhimurium and L. monocytogenes in raw beef. LAMP bioluminescence assays provide results on the subsequent day and are simple to use compared with the Korea Standard Food Codex (KFSC) Method, particularly in terms of DNA preparation.

Detection of Pathogenic Yersinia Enterocolitica in Drinking Water and Vegetables by Mutiplex-PCR (Multiplex-PCR에 의한 먹는샘물 및 야채류로부터의 병원성 Yersinia enterocolitica의 신속검출)

  • 이택수;박부길;오덕환
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.32 no.1
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    • pp.35-41
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    • 2003
  • The study was conducted to develope a rapid method for the detection of Yersinia enterocolitica in spring water and vegetables via multiplex polymerase chain reaction (PCR) technique using ail, yst, uirF and subgenus-specific Y16S primers. Specificity and sensitivity of multiplex PCR and application of best primers for the detection of Y. enterocolitica from spring water and vegetables were investigeted. Y. enterocolitica ATCC 27729 strains gave 356 bP and 200 bp (Y16S) and 134 bp (yst) bands. but Y. enterocolitica ATCC 9610 and ATCC 23715 strains gave 200 bp and 134 bp bands.In the meanwhile, non-pathogenic Yersinia species, such as Y. frederikseni, Y. inter-media, Y. kristenseni and Y. pseudotuberculosis gave only single 200 bp band, and other bacteria including Escherichia coli O157:H7 ATCC 25392, Shigella dysenteri. Staphylococcu aureus ATCC 25923 and Listeria mo-nocytogenes ATCC 19111 did not show any bands. Among primers, yst and Y16S primer showed the best sensitivity. Seven CFU/mL Y. enterocolitica cells could be detected with yst and Y16S primers and the sensitivity was significantly improved by the further 2nd PCR after 38 cycles of first PCR amplication. Spring water, cabbage and mushroom were inoculated with Y. enterocolitica to determine the sensitivity of multiplex-PCR for the rapid detection of Y. enterocolitica. Multiplex-PCR assay could detect 7 or 70 cells in spring water and vegetables using whole cell lysate with repeating PCR amplication.

Analysis of Vitellogenin Gene Expression in Synechogobius hastus (Gobiidae) (풀망둑 난황전구단백질 유전자발현 추적기법)

  • 계명찬
    • Korean Journal of Environmental Biology
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    • v.22 no.1
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    • pp.206-212
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    • 2004
  • In an effort to develop the tools for monitoring the contamination of xenoestrogen in the aquatic environment of Korea, reverse transcription-polymerase chain reaction (RT-PCR) analysis of vitellogenin (VTG) mRNA expression were optimized in Synechogobius hastus. Based on the partial VTG cDNA sequence VTG mRNA level in livers from male fishes was analyzed by RT-PCR. As an internal control beta actin mRNA was amplified. 3 ${\mu}g$ of total RNA was reverse transcribed in 20 $\mu$l reaction using murine leukemia virus 〔MuLV〕 reverse transcriptase. Subsequent PCR using the 1 ${\mu}g$ of cDNA resulted in linear increase in PCR product of VTG in female liver cDNA from 10 to 30 cycles of amplification. On the contrary, in male, PCR product first detected at 28 cycles of amplification and linearly increased during 38 cycles of amplification, suggesting that male S. hastus expresses minute amount of VTG mRNA which is $2^{-18}$ equivalent of female. In conclusion, the optimized protocol of VTG mRNA expression in the liver of male S. hastus will be promising the environmental monitoring the xenoestrogen contamination in the western coast and estuaries in Korea.