共查询到20条相似文献,搜索用时 15 毫秒
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G. JENÍKOVÁ A.N. JENSEN K. DEMNEROVÁ J. HOORFAR 《Journal of Rapid Methods and Automation in Microbiology》2001,9(3):135-141
Four rapid and simple DNA purification and sample treatment protocols were evaluated for detection of Salmonella enterica in spiked minced meat, using a fluorogenic 5'nuclease (TaqMan) PCR assay in an ABI-Prism 7700 Sequence Detector. The detection limit with the single separation treatment of DNeasy was found to be 6–8 CFU in just 19 end-point fluorescence (Ct ) values, while this was 22 Ct for a combination of DNeasy and BactXtractor. Extraction by DNeasy resulted in Ct <22, indicating a detection limit of <10 Salmonella cells per 25g, when the samples were inoculated with Salmonella before the overnight preenrichment. The method is currently being adapted to a BioRobot 3000 platform. However, the use of paramagnetic beads (DNA Direct) resulted in poor and variable detection limit. 相似文献
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BARBARA J. JACKSON ALISA M. BROOKINS DONNA TETREAULT KATHERINE COSTELLO 《Journal of Rapid Methods and Automation in Microbiology》1993,2(1):39-54
Detection of Listeria by capture on antibody-coated magnetic beads has been shown to decrease test time and improve sensitivity, relative to cultural methods, in a study of spiked environmental samples (Mitchell et al. 1993). In this study, immunomagnetic capture was compared to standard cultural methods for detection of Listeria in a broad range of spiked and naturally contaminated food and environmental samples. Immunomagnetic capture was at least as sensitive as cultural methods for detection of Listeria in seafood, meats, dairy foods, and environmental samples. It was possible to determine the number of Listeria present in a sample, because immunomagnetic capture was carried out directly from the sample, without enrichment. These quantitative results were produced within 24 h, while cultural methods required 6–14 days to produce a qualitative result. Immunomagnetic capture was thus more rapid and as sensitive as standard cultural methods for detection of Listeria in the food and environmental samples tested. 相似文献
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DONG-SHENG ZHU MIN ZHOU YI-LING FAN XIAN-MING SHI 《Journal of Rapid Methods and Automation in Microbiology》2009,17(1):67-79
A genome comparison method was used to identify specific target sequences for the polymerase chain reaction (PCR) detection of Vibrio parahaemolyticus, and the CDS value of this bacterium was compared with that of 139 other bacterial genomes. It was found that 20 CDS of V. parahaemolyticus were relatively specific according to their E value in BLAST (a new tool for comparing protein and nucleotide sequences), and four of them were selected for the design of PCR primers. There were positive amplification products of these four pairs of primers from nine V. parahaemolyticus strains, whereas there were no amplification products from nine other Vibrionaceae strains and four non -Vibrionaceae strains. An evaluation of detection sensitivities revealed that these four pairs of primers can be used in a PCR assay for the detection of V. parahaemolyticus.
An automatic BLAST method was developed in this study, by which species-specific sequences can be screened out rapidly. In this way, new and specific genes of Vibrio parahaemolyticus were identified to be used as target sequences for PCR detection. In terms of acceptable specificity and sensitivity, the four pairs of primers were selected by screening, which can be applied in PCR assays and other molecular methods. These kinds of methods might become commercial detection products in the new future. In addition, this method for searching specific DNA sequences can also be used for the mining specific sequences in other genus and species, such as Salmonella , Staphylococcus , etc. 相似文献
PRACTICAL APPLICATIONS
An automatic BLAST method was developed in this study, by which species-specific sequences can be screened out rapidly. In this way, new and specific genes of Vibrio parahaemolyticus were identified to be used as target sequences for PCR detection. In terms of acceptable specificity and sensitivity, the four pairs of primers were selected by screening, which can be applied in PCR assays and other molecular methods. These kinds of methods might become commercial detection products in the new future. In addition, this method for searching specific DNA sequences can also be used for the mining specific sequences in other genus and species, such as Salmonella , Staphylococcus , etc. 相似文献
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应用PCR技术检测病人血液标本中斑点热群立克次体DNA 总被引:1,自引:1,他引:1
本文以聚合酶链反应(PCR),采用二次扩增法直接检测蜱传斑点热病人血液标本中斑点热群立克次体DNA,结果从7份标本中检出阳性1份,进一步的限制性片段多态性分析证明和斑点热群黑龙江立克次体基因型相同。实验证明:黑龙江立克次体对人具有致病性,同时也证明PCR技术快速、简单、敏感,用于斑点热群立克次体的早期诊断是可行的,并可作出分型鉴定。 相似文献
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KHADIJA IBENYASSINE RAJAA AITMHAND YAHAYA KARAMOKO MOULAY MUSTAPHA ENNAJI 《Journal of Rapid Methods and Automation in Microbiology》2008,16(2):113-121
Contaminated vegetables have been identified as one of the principal sources of foodborne illnesses. Escherichia coli is one of the bacteria that can contaminate vegetables and cause serious foodborne disease. The development of simple and rapid assays for detection of E. coli would enable official agencies and food industries to identify contaminated foodstuffs in a timelier manner. In this work, we detected E. coli contamination in four types of vegetables using a 24 h procedure. This method is very specific, rapid and simple to use in the laboratory. Indeed, the enrichment, DNA isolation and polymerase chain reaction (PCR) amplification procedures described here can also be used for detection of E. coli in other foods.
Foodborne disease remains an important public health threat worldwide; one of the most important food safety hazards is associated with raw vegetables. Several studies have found that products can support the growth of enteric bacterial pathogens such as Salmonella , Shigella and Escherichia coli . Culture techniques are universally recognized as the standard method for detecting pathogenic bacteria in foods. Bacteria are detected and subsequently identified by growth on solid selective culture media and by analysis of metabolic properties or serotyping. This process is lengthy and may last 5–10 days or more. In our investigation, the polymerase chain reaction (PCR) detection of E. coli in vegetables was realized within 24 h. The pre-enrichment step used in this study did not have any inhibiting effect on the PCR. Therefore, it became possible to rapidly and directly detect E. coli in raw vegetables by the PCR technique just by heating samples. 相似文献
PRACTICAL APPLICATIONS
Foodborne disease remains an important public health threat worldwide; one of the most important food safety hazards is associated with raw vegetables. Several studies have found that products can support the growth of enteric bacterial pathogens such as Salmonella , Shigella and Escherichia coli . Culture techniques are universally recognized as the standard method for detecting pathogenic bacteria in foods. Bacteria are detected and subsequently identified by growth on solid selective culture media and by analysis of metabolic properties or serotyping. This process is lengthy and may last 5–10 days or more. In our investigation, the polymerase chain reaction (PCR) detection of E. coli in vegetables was realized within 24 h. The pre-enrichment step used in this study did not have any inhibiting effect on the PCR. Therefore, it became possible to rapidly and directly detect E. coli in raw vegetables by the PCR technique just by heating samples. 相似文献
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鱼类病原菌中R^+质粒的检出及其特性 总被引:3,自引:0,他引:3
本文研究了5个属的15株鱼类病原菌对17种抗菌药物的耐药性,其中,对β-内酰胺类和红霉素的耐药率最高,对妥霉素最敏感。以E.coliK-12RC85为受体菌,鱼类病原菌为供体菌,采用细菌接合试验,从耐氨苄青霉素,四环素和磺胺的嗜水气单胞菌CJ26株中检测到一株耐四环素和磺和磺胺的可自身传递的R质粒PWH9601。它对四环素的抗性可被四环素类药制所诱导。PWH9601上的耐药基因在供体菌和受体菌中的 相似文献
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RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION 总被引:1,自引:0,他引:1
LIJU YANG CHUANMIN RUAN YANBIN LI 《Journal of Rapid Methods and Automation in Microbiology》2001,9(4):229-240
A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 103 105 cfu/mL, with R2 = 0.99. The detection limit of this method was 1.09 × 103 cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples. 相似文献
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RT套式PCR检测血浆HCV RNA及与抗HCV检测的比较 总被引:7,自引:0,他引:7
应用微量血清热变性法提取核酸,逆转录套式聚合酶链反应(RT-nest PCR)检测血浆HCV RNA,并与抗HCV ELISA检测结果比较,对HCV RNA阳性标本进行HGV RNA的筛查.结果在32例抗HCV阳性和20例抗HCV阴性血浆中,HCV RNA分别检出18例和2例,总符合率为70%,20例HCV RNA阳性者中有2例合并感染HBV,1例合并感染HGV.证明血浆样本中抗HCV与HCV RNA间存在很大的相关性. 相似文献
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用高效液相色谱—电化学检测法快速、灵敏分析生物样本中的单胺类递质及其代谢产物 总被引:16,自引:0,他引:16
本实验用反相离子对色谱-电化学检测分析法,同时分离并测定生物样本中结构极为相似的单胺类递质及其代谢产物。实验采用等比洗脱,在30min内至少可测定九个这类化合物。生物样品的预处理简单,因而平均回收率能达到90%以上。十个化合物(包括3,4-二羟基苄胺)保留时间变异系数为0.73±0.18%(均值±标准差,下同);峰面积变异系数为1.11±0.45%;在50—100pg至40ng范围内,相夫系数为0.996±0.006。本文对我们所用的色谱分析条件进行了讨论。 相似文献
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PCR直接检测龈下菌斑主要可疑牙周致病菌 总被引:11,自引:0,他引:11
目的:应用PCR方法直接检测龈下菌斑主要可疑牙周致病菌与牙周病活动部位的关系,探讨其方法的可行性并探讨其主要可疑牙周致病菌的分布规律。方法:应用聚合酶链反应(polymerase chain reaction,PCR)直接检测龈下菌斑主要可疑致病菌16s RNA保守区域片段。40名受试者包括牙周病患者20人,每人同口取一个牙周病活动部位,一个相对健康或牙周病静止对照部位;成人健康者20人,每人各取一个标本。结果:龈下菌斑5种可疑牙周致病菌在牙周病活动部位的检出率牙龈卟啉菌为86%,福赛类杆菌为95%,螺旋体为86%,中间普氏菌和黑色普氏菌分别为95%和33%,均显著高于同口部位对照组和健康对照组。结论:PCR直接检测菌斑牙龈卟啉单胞菌、中间普氏菌、福赛类杆菌、齿密螺旋体及黑色普氏菌匀与牙周炎活动部位相关。 相似文献
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Christopher A. Scholin Roman Marin III Peter E. Miller Gregory J. Doucette Christine L. Powell Paul Haydock Judith Howard Jason Ray 《Journal of phycology》1999,35(6):1356-1367
Large-subunit ribosomal RNA-targeted probes for Pseudo-nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole-cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996–1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole-cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor-binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis cross-reacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole-cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells. 相似文献
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A fluorometric technique, based on the combination of RNase and DNase incubation with the use of thiazole orange (RNase/DNase method), was investigated to determine DNA and RNA concentrations in marine plankton. Tests were performed to optimize both RNase and DNase assay conditions. The RNase assay should be conducted at 37° C for 20 min with 0.5 μg·mL?1 of DNase-free RNase. An incubation at 25° C for 20 min with 10 units ·mL-1 of RNase-free DNase were the optimal conditions required for DNA digestion by DNase. The detection limits in terms of minimum biomass for reliable measurements of DNA and RNA were 7.5 and 10 μg of protein · (mL assay)?1, respectively. RNA and DNA concentration were estimated in oligotrophic water samples using the RNase/DNase and other available methods (e.g. a double fluorochrome method). The different techniques provided similar DNA estimations. However, the RNase/DNase method provided the highest sensitivity and a low variability for the estimation of RNA. 相似文献