A modified low denaturing temperature PCR (LDT-PCR) method combined with DNA microarray technique is developed in our lab for quick and effective identification of various mutations in an 81 base pair region of Mycoba...A modified low denaturing temperature PCR (LDT-PCR) method combined with DNA microarray technique is developed in our lab for quick and effective identification of various mutations in an 81 base pair region of Mycobacterium Tuberculosis (MTB) ribosome RNA polymerase subunit B (rpoB) gene associated with rifampin resistance. By incurporation of wild type (wt) allele fragments that had been PCR amplified previously, the target PCR fragments coming from mutant clinical MTB samples were codenaturized with incorporated wt type allele fragment at 94°C and then let them randomly form matched structures (homoduplex) and allele mismatch-containing structures (heteroduplex), respectively, when the temperature cooled down to 70°C. After the temperature was raised to 80°C, the heteroduplex double stranded fragments were preferentially denatured and resulted in PCR amplification as well as fluorescence incurporation. Since the homoduplex fragments need a higher temperature to be denatured, they were kept in double-stranded status at that temperature and failed to be PCR amplified. By hybridization of LDT-PCR products with the probes spotted on microarray slides, the fluorescent signals representing the presence of gene mutations were detected. We have tested this method on 35 clinical MTB samples and obtained satisfied results.展开更多
Axle load data are an essential input for pavement design,yet for most North American agencies,there is uncertainty about the quality of axle load data obtained from weigh-inmotion(WIM)systems,the applicability of the...Axle load data are an essential input for pavement design,yet for most North American agencies,there is uncertainty about the quality of axle load data obtained from weigh-inmotion(WIM)systems,the applicability of these data for pavement design,and potential opportunities to integrate axle load data from disparate sources.This article presents a novel and practical methodology to evaluate the quality of axle load data from WIM systems and roadside weigh scales through a series of hierarchical analyses designed to test data validity.When applied using data from Manitoba,Canada,the methodology quantified the uncertainty of axle loads measured at the weigh scales and piezo-quartz WIM,concluding that both could be used for pavement design applications.Data collected at piezo-polymer WIM sites exhibited poorer data validity;however,application of site-specific temperature correction factors significantly improved data validity at these sites.The article describes how other data quality dimensions,including spatial coverage,temporal coverage,and long-term data availability,could be considered when determining the suitability of disparate axle load data sources for pavement design.Application of the methodology enables a pragmatic evaluation of the quality and limitations of commonlyavailable axle load data,revealing uncertainties and data needs relevant for pavement design practice.展开更多
文摘A modified low denaturing temperature PCR (LDT-PCR) method combined with DNA microarray technique is developed in our lab for quick and effective identification of various mutations in an 81 base pair region of Mycobacterium Tuberculosis (MTB) ribosome RNA polymerase subunit B (rpoB) gene associated with rifampin resistance. By incurporation of wild type (wt) allele fragments that had been PCR amplified previously, the target PCR fragments coming from mutant clinical MTB samples were codenaturized with incorporated wt type allele fragment at 94°C and then let them randomly form matched structures (homoduplex) and allele mismatch-containing structures (heteroduplex), respectively, when the temperature cooled down to 70°C. After the temperature was raised to 80°C, the heteroduplex double stranded fragments were preferentially denatured and resulted in PCR amplification as well as fluorescence incurporation. Since the homoduplex fragments need a higher temperature to be denatured, they were kept in double-stranded status at that temperature and failed to be PCR amplified. By hybridization of LDT-PCR products with the probes spotted on microarray slides, the fluorescent signals representing the presence of gene mutations were detected. We have tested this method on 35 clinical MTB samples and obtained satisfied results.
基金financial contributions of Manitoba Infrastructure and the Natural Sciences and Engineering Research Council(NSERC)of Canada(grant number RGPIN/418427-2012)。
文摘Axle load data are an essential input for pavement design,yet for most North American agencies,there is uncertainty about the quality of axle load data obtained from weigh-inmotion(WIM)systems,the applicability of these data for pavement design,and potential opportunities to integrate axle load data from disparate sources.This article presents a novel and practical methodology to evaluate the quality of axle load data from WIM systems and roadside weigh scales through a series of hierarchical analyses designed to test data validity.When applied using data from Manitoba,Canada,the methodology quantified the uncertainty of axle loads measured at the weigh scales and piezo-quartz WIM,concluding that both could be used for pavement design applications.Data collected at piezo-polymer WIM sites exhibited poorer data validity;however,application of site-specific temperature correction factors significantly improved data validity at these sites.The article describes how other data quality dimensions,including spatial coverage,temporal coverage,and long-term data availability,could be considered when determining the suitability of disparate axle load data sources for pavement design.Application of the methodology enables a pragmatic evaluation of the quality and limitations of commonlyavailable axle load data,revealing uncertainties and data needs relevant for pavement design practice.
