A PCR IMMUNOASSAY METHOD FOR THE DETECTION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES

PCR primers targeting the internal transcribed spacer (ITS)-5.8S rDNA regions specific for the genus Alexandrium were used to develop an ELISA assay method to detect and enumerate this genus in cultured isolates. The solid-phase ELISA involves the application of a biotinylated labeled primer to target the specific ITS-5.8S rDNA region; the PCR-amplified products, generated in the presence of digoxigenin-11-deoxiuracil triphosphate nucleotide, are captured on the streptavidin-coated microplate. The captured molecules were hybridized to an anti-digoxigenin antibody conjugated with alkaline phosphatase. The presence and number of the Alexandrium cells in the samples resulted in a proportional appearance of color generated by the phosphatase activity in the presence of a chromogenic substrate and measured in a plate reader. This PCR and immunoassay solid-phase assay proved to be a useful technique to detect the presence of Alexandrium sp. in cultured isolates and seawater samples.     

A SIMPLIFIED METHOD FOR THE PREPARATION OF EGG YOLK MEDIA

SUMMARY: Difficulties with filtration in the preparation of egg yolk media were overcome by preparing the yolk emulsion with distilled water instead of saline. The turbidity of the final medium was related to its sodium chloride content.     

MINIATURIZED ANALYTICAL METHOD FOR THE DETECTION OF AMINE PRODUCTION BY MICROORGANISMS

Histamine, the result of histidine decarboxylation, has been associated with allergic reactions due to the consumption of certain foods. Other biogenic amines, such as putrescine and cadaverine, have been related to quality deterioration in foods. A quantitative miniaturized method for the detection of biogenic amines produced by microorganisms in culture media, was designed. The reaction takes place in microplates containing microquantities of inoculated media and reagents. Amine production is determined spectrophotometrically by monitoring changes in the acid phase of the pH indicator at 405 nm. Using the following amino acids: histidine, phenylalanine, tyrosine, tryptophan, arginine, lysine and ornithine, 44 microorganisms were tested for amine production. Sensitivity of the method is 10 μM of amine.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

一种cDNA克隆的简易方法

构建了一种适用于克隆cDNA的双分子质粒载体。使用此载体,以载体引物合成ds-cDNA后,用连接酶将cDNA-载体重组子自身环化,转化宿主菌。本文以此方法构建了人胚胎组织cDNA文库,转化菌中约50％含有cDNA插入片段。此方法大大简化了cDNA克隆步骤,提高了克隆效率。     

一种用分子杂交技术检测谷氨酸生产菌溶原性的方法

谷氨酸生产菌用于发酵生产谷氨酸和其他氨基酸,由于外源噬菌体污染或者由溶原菌内诱导出的噬菌体污染,往往给工业发酵带来严重的危害,于是测定谷氨酸生产菌是否为溶原菌便成了当务之急。本文以溶原菌E.coli K_(12)为研究模型,首先用~(32)P标记的λDNA作探针,建立了检测溶原菌株的分子诊断方法,继而通过寄主菌株从生产环境样品中分离筛选到7_6和7_(?)两大类共20多株噬菌体。由该两类噬菌体制得的~(32)P-7_(?)和7_(?)探针,可分别与以生产谷氨酸的棒杆菌B_9、7338和T_(?)三个菌株为寄主,从环境样品中分离的18个和6个噬菌体分离物杂交。而采用缺口翻译系统制备的7_(?)和7_(?)混合探针,或混合使用7_(?)和(?)探针,使得检测方法更为简便易行。通过Southern Blot技术和菌落杂交对菌DNA的分析,结果表明三个寄主菌株均不是所分离的噬菌体的溶原菌。最后,本文还就烈性噬菌体和温和噬菌体之间的同源性与分子诊断的可行性作了初步探讨。     

A SIMPLIFIED METHOD FOR THE URINARY EXCRETION TEST OF ABSORPTION OF COBALT 60 LABELLED VITAMIN B12

It is possible to do both radioiodine uptake studies and urinary excretion tests for absorption of Cobalt 60 vitamin B₁₂ with the same basic equipment. If a spectrometer is used and the samples properly prepared, the latter procedure can be performed quickly, accurately and with a minimum of laboratory equipment. This is a distinct advantage to a small laboratory with limited facilities, and should help to make both of these useful tests available for clinical use.     

AN ELECTRICAL TESTING METHOD FOR THE DETECTION OF LISTERIA SPECIES IN CONFECTIONERY PRODUCTS

An electrical testing method for the detection of Listeria spp. in confectionery products and associated raw materials was developed in which samples can be negatively screened within 48h. The method involves a 24h resuscitation in Listeria enrichment broth followed by a 24h test in a Bactometer M128 using a broth (Claremont broth) developed from Oxford agar. The comparative study involved analysis of 511 samples (chocolate, dairy, cereal, nut and fruit products) tested by the Interim Australian Standard method (AS) and the Bactometer method (BM). The sensitivity and specificity of the BM for samples §1 Listeria/g was 100% and 99.8%, respectively, when compared to the AS method. When samples containing < 1 Listeria/g sample were added to the analysis, the sensitivity and specificity marginally dropped (98.3% and 99.6%). The electrical capacitance method is rapid and easy to perform, with a negative result being available within 48h making it a viable tool in a positive release QA program in food manufacturing factories.