Listeria monocytogenes is an important foodborne pathogen responsible for listeriosis,a fatal disease.It is widely distributed in various foods and environmental sources.In this review,we focused on addressing PCR-bas...Listeria monocytogenes is an important foodborne pathogen responsible for listeriosis,a fatal disease.It is widely distributed in various foods and environmental sources.In this review,we focused on addressing PCR-based technologies,including conventional PCR,qPCR and droplet digital PCR(ddPCR).Specifically,we described(a)conventional PCR and mono-,duplex-and multiplex-qPCR methodologies;(b)development and applications of gene HlyA-,Iap-,PrfA–and SsrA-based conventional and qPCR assays as well as PCR assays targeting newly identified gene targets for specific detection of L.monocytogenes;differentiation of viable from dead L.monocytogenes by qPCR in conjugation with propidium monoazide pretreatment;PCR-based serotype identification of L.monocytogenes isolates;PCR-based detection of L.ivanovii,infecting ruminants,differentiation of L.monocytogenes from other Listeria species;and sigB-gene based PCR identification of Listeria spp;(c)applications of ddPCR in detection of L.monocytogenes;and(d)application of qPCR assays in detection and subtyping of L.monocytogenes in milk and dairy products;meats,meat products and meat-processing environment;and seafood,seafood products and processing environment.Our goal was to provide a relatively comprehensive overview of PCR-based methodologies available in detection,characterization and subtyping of various strains of L.monocytogenes in foods and environmental sources.展开更多
Listeria monocytogenes,one of the most important foodborne pathogens,can cause listeriosis,a lethal disease for humans.L.ivanovii,which is closely related to L.monocytogenes,is also widely distributed in nature and in...Listeria monocytogenes,one of the most important foodborne pathogens,can cause listeriosis,a lethal disease for humans.L.ivanovii,which is closely related to L.monocytogenes,is also widely distributed in nature and infects mainly warm-blooded ruminants,causing economic loss.Thus,there are high priority needs for methodologies for rapid,specific,cost-effective and accurate detection,characterization and subtyping of L.monocytogenes and L.ivanovii in foods and environmental sources.In this review,we(A)described L.monocytogenes and L.ivanovii,world-wide incidence of listeriosis,and prevalence of various L.monocytogenes strains in food and environmental sources;(B)comprehensively reviewed different types of traditional and newly developed methodologies,including culture-based,antigen/antibody-based,LOOP-mediated isothermal amplification,matrix-assisted laser desorption ionization-time of flight-mass spectrometry,DNA microarray,and genomic sequencing for detection and characterization of L.monocytogenes in foods and environmental sources;(C)comprehensively summarized different subtyping methodologies,including pulsed-field gel electrophoresis,multi-locus sequence typing,ribotyping,and phage-typing,and whole genomic sequencing etc.for subtyping of L.monocytogenes strains from food and environmental sources;and(D)described the applications of these methodologies in detection and subtyping of L.monocytogenes in foods and food processing facilities.展开更多
Shiga toxin producing Escherichia coli(STEC)outbreak is a public health concern as it can potentially cause a variety of clinical manifestations including diarrhea,hemorrhagic colitis and hemolytic uremic syndrome(HUS...Shiga toxin producing Escherichia coli(STEC)outbreak is a public health concern as it can potentially cause a variety of clinical manifestations including diarrhea,hemorrhagic colitis and hemolytic uremic syndrome(HUS).However E.coli are generally innocuous commensal organisms,and there is a need to discriminate pathogenic from non-pathogenic isolates rapidly and accurately.In this study,we have used standard culture based methods and advanced molecular approaches to characterize E.coli in food in a local outbreak investigation.We show that the application of DNA based detection methods including real-time PCR and DNA microarray along with a traditional culture method can identify the organism implicated in an outbreak at the strain level for pathogenic potential.展开更多
The development and implementation of advanced analytical technologies is essential for extending the expiry for complex drug products stored in the Strategic National Stockpiles. Consequently, a novel Ultra High-Perf...The development and implementation of advanced analytical technologies is essential for extending the expiry for complex drug products stored in the Strategic National Stockpiles. Consequently, a novel Ultra High-Performance Liquid Chromatographic (UHPLC) method has been developed for the analysis of atropine and its respective impurities to support the analytical research platform for auto-injectors. This study is part of a larger research effort to improve the efficiency and broaden the applicability of advanced analytical methods for medical counter-measure medications. The current HPLC compendial methodology for atropine sulfate injection requires an analysis time of 40 minutes for atropine. In comparison, the novel gradient UHPLC method required only 8 minutes to evaluate both atropine and its major pharmaceutical impurities. Improved separation was achieved on a Waters Acquity UHPLC BEH C18 1.7 μm, 2.1 × 100 mm column employing gradient elution of mobile phase solvent A (0.1% H3PO4) and solvent B (0.1% H3PO4, 90% ACN, and 10% H2O). The method was validated according to USP Category I requirements for assay. The daily standard calibration curves were linear over a concentration range from 50 μg/mL to 250 μg/mL with a correlation coefficient of >0.999. The detection limit (LOD) and quantitation limit (LOQ) were 3.9 μg/ml and 13.1 μg/ml, respectively. Resolution results indicate that atropine and the following impurities, degradants and a preservative can also be separated and analyzed using this proposed method: noratropine, 4,4’-di-hy-droxydiphenyl ether, 2,4’-dihydroxydiphenyl ether, 4-bromophenol, 4-hydro-xyatropine, tropic acid, apoatropine HCl, atropic acid, hydroquinone, nitroethane, phenol and catechol. The UHPLC method demonstrated enhanced selectivity and significantly reduced the analysis time when compared with the traditional USP compendial HPLC method. The method was successfully applied to the evaluation of atropine in ATNAA auto-injectors lots from the Strategic National Stockpiles.展开更多
文摘Listeria monocytogenes is an important foodborne pathogen responsible for listeriosis,a fatal disease.It is widely distributed in various foods and environmental sources.In this review,we focused on addressing PCR-based technologies,including conventional PCR,qPCR and droplet digital PCR(ddPCR).Specifically,we described(a)conventional PCR and mono-,duplex-and multiplex-qPCR methodologies;(b)development and applications of gene HlyA-,Iap-,PrfA–and SsrA-based conventional and qPCR assays as well as PCR assays targeting newly identified gene targets for specific detection of L.monocytogenes;differentiation of viable from dead L.monocytogenes by qPCR in conjugation with propidium monoazide pretreatment;PCR-based serotype identification of L.monocytogenes isolates;PCR-based detection of L.ivanovii,infecting ruminants,differentiation of L.monocytogenes from other Listeria species;and sigB-gene based PCR identification of Listeria spp;(c)applications of ddPCR in detection of L.monocytogenes;and(d)application of qPCR assays in detection and subtyping of L.monocytogenes in milk and dairy products;meats,meat products and meat-processing environment;and seafood,seafood products and processing environment.Our goal was to provide a relatively comprehensive overview of PCR-based methodologies available in detection,characterization and subtyping of various strains of L.monocytogenes in foods and environmental sources.
文摘Listeria monocytogenes,one of the most important foodborne pathogens,can cause listeriosis,a lethal disease for humans.L.ivanovii,which is closely related to L.monocytogenes,is also widely distributed in nature and infects mainly warm-blooded ruminants,causing economic loss.Thus,there are high priority needs for methodologies for rapid,specific,cost-effective and accurate detection,characterization and subtyping of L.monocytogenes and L.ivanovii in foods and environmental sources.In this review,we(A)described L.monocytogenes and L.ivanovii,world-wide incidence of listeriosis,and prevalence of various L.monocytogenes strains in food and environmental sources;(B)comprehensively reviewed different types of traditional and newly developed methodologies,including culture-based,antigen/antibody-based,LOOP-mediated isothermal amplification,matrix-assisted laser desorption ionization-time of flight-mass spectrometry,DNA microarray,and genomic sequencing for detection and characterization of L.monocytogenes in foods and environmental sources;(C)comprehensively summarized different subtyping methodologies,including pulsed-field gel electrophoresis,multi-locus sequence typing,ribotyping,and phage-typing,and whole genomic sequencing etc.for subtyping of L.monocytogenes strains from food and environmental sources;and(D)described the applications of these methodologies in detection and subtyping of L.monocytogenes in foods and food processing facilities.
文摘Shiga toxin producing Escherichia coli(STEC)outbreak is a public health concern as it can potentially cause a variety of clinical manifestations including diarrhea,hemorrhagic colitis and hemolytic uremic syndrome(HUS).However E.coli are generally innocuous commensal organisms,and there is a need to discriminate pathogenic from non-pathogenic isolates rapidly and accurately.In this study,we have used standard culture based methods and advanced molecular approaches to characterize E.coli in food in a local outbreak investigation.We show that the application of DNA based detection methods including real-time PCR and DNA microarray along with a traditional culture method can identify the organism implicated in an outbreak at the strain level for pathogenic potential.
文摘The development and implementation of advanced analytical technologies is essential for extending the expiry for complex drug products stored in the Strategic National Stockpiles. Consequently, a novel Ultra High-Performance Liquid Chromatographic (UHPLC) method has been developed for the analysis of atropine and its respective impurities to support the analytical research platform for auto-injectors. This study is part of a larger research effort to improve the efficiency and broaden the applicability of advanced analytical methods for medical counter-measure medications. The current HPLC compendial methodology for atropine sulfate injection requires an analysis time of 40 minutes for atropine. In comparison, the novel gradient UHPLC method required only 8 minutes to evaluate both atropine and its major pharmaceutical impurities. Improved separation was achieved on a Waters Acquity UHPLC BEH C18 1.7 μm, 2.1 × 100 mm column employing gradient elution of mobile phase solvent A (0.1% H3PO4) and solvent B (0.1% H3PO4, 90% ACN, and 10% H2O). The method was validated according to USP Category I requirements for assay. The daily standard calibration curves were linear over a concentration range from 50 μg/mL to 250 μg/mL with a correlation coefficient of >0.999. The detection limit (LOD) and quantitation limit (LOQ) were 3.9 μg/ml and 13.1 μg/ml, respectively. Resolution results indicate that atropine and the following impurities, degradants and a preservative can also be separated and analyzed using this proposed method: noratropine, 4,4’-di-hy-droxydiphenyl ether, 2,4’-dihydroxydiphenyl ether, 4-bromophenol, 4-hydro-xyatropine, tropic acid, apoatropine HCl, atropic acid, hydroquinone, nitroethane, phenol and catechol. The UHPLC method demonstrated enhanced selectivity and significantly reduced the analysis time when compared with the traditional USP compendial HPLC method. The method was successfully applied to the evaluation of atropine in ATNAA auto-injectors lots from the Strategic National Stockpiles.
