The Experts below are selected from a list of 115392 Experts worldwide ranked by ideXlab platform
Hirotaka Konuma - One of the best experts on this subject based on the ideXlab platform.
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development of a quantitative real time polymerase chain reaction targeted to the toxr for detection of vibrio vulnificus
Journal of Microbiological Methods, 2005Co-Authors: Hajime Takahashi, Yukiko Harakudo, Jiro Miyasaka, Susumu Kumagai, Hirotaka KonumaAbstract:Abstract The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene ( toxR ) of Vibrio vulnificus . The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus . We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.
Toyo Okui - One of the best experts on this subject based on the ideXlab platform.
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Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription-polymerase chain reaction.
Journal of medical virology, 2008Co-Authors: Setsuko Ishida, Shima Yoshizumi, Tetsuya Ikeda, Masahiro Miyoshi, Motohiko Okano, Toyo OkuiAbstract:Conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect norovirus (NV) is a complex of multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT-PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1-ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT-PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT-PCR. Two hundred fifty-one of the 255 specimens that were negative by the conventional RT-PCR were also negative by the TaqMan RT-PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT-PCR end-point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT-PCR is useful for the detection of NV in clinical specimens. J. Med. Virol. 80:913–920, 2008. © 2008 Wiley-Liss, Inc.
Mike G Makrigiorgos - One of the best experts on this subject based on the ideXlab platform.
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coamplification at lower denaturation temperature pcr increases mutation detection selectivity of TaqMan based real time pcr
Clinical Chemistry, 2009Co-Authors: Jin Li, Lilin Wang, Pasi A Janne, Mike G MakrigiorgosAbstract:Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.
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coamplification at lower denaturation temperature pcr increases mutation detection selectivity of TaqMan based real time pcr
Clinical Chemistry, 2009Co-Authors: Lili Wang, Pasi A Janne, Mike G MakrigiorgosAbstract:Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature ( T c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.
Gitika Panicker - One of the best experts on this subject based on the ideXlab platform.
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real time pcr detection of vibrio vulnificus in oysters comparison of oligonucleotide primers and probes targeting vvha
Applied and Environmental Microbiology, 2005Co-Authors: Gitika PanickerAbstract:We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (CT) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103 V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 103 CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.
Hajime Takahashi - One of the best experts on this subject based on the ideXlab platform.
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development of a quantitative real time polymerase chain reaction targeted to the toxr for detection of vibrio vulnificus
Journal of Microbiological Methods, 2005Co-Authors: Hajime Takahashi, Yukiko Harakudo, Jiro Miyasaka, Susumu Kumagai, Hirotaka KonumaAbstract:Abstract The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene ( toxR ) of Vibrio vulnificus . The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus . We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.
