Bioluminescent methods of analysis in microbiology

The prospects for application of bioluminescent ATP-metry in microbiology are considered. A bioluminescent assay is proposed to analyse biomass by measuring the content of intracellular ATP by means of immobilized firefly luciferase after ATP extraction with dimethylsulfoxide. The assay can be used for plotting the growth curves of microorganisms and for determining the sensitivity of microorganisms to antibiotics. The detection limit of the assay is 700 cells per ml of the measured solution.     

不同方法检测鸡蛋表面菌落总数的相关性研究

研究了ATP生物荧光检测法与国标法《GB4789.2-2010食品卫生微生物学检验菌落总数测定》检测鸡蛋壳表面细菌总数的相关性。采用ATP生物荧光检测法和国标法对40个混合样品表面细菌总数进行检测,以log CFU/个蛋壳为横坐标(x),以logRLU/个蛋壳为纵坐标(y),分别进行线性、对数、乘幂、指数拟合。结果表明,ATP荧光检测法与国标法检测结果 Pearson相关系数为0.912,线性模型y=0.7306x-1.0041(R2=0.8322)拟合度较高。该试验结果为ATP荧光检测法在鸡蛋壳表面细菌总数快速检测中应用的可行性提供了依据。     

Rapid microbiology: Applications of bioluminescence in the food industry a review

The bioluminescent assay of ATP is rapid and simple and may be used as an estimate of microbial numbers. It therefore shows great potential as a technique to provied information on the microbiological quality of a food within a few minutes, in comparison with conventional techniques, which provide results retrospectively. Howver, despite the advantages of speed and senstitivity, no food microbiologists are using the technique for routine quality control and hygiene monitoring. This review seeks to highlight the reasons for this, and to offer some ideas for future research to increase the acceptance of ATP assays within the food industry.     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples

A recent PCR detection technique (TaqMan) based on the 5'-3'-exonuclease activity of the Taq DNA polymerase was applied to the detection of indicator organisms in water samples. In this technique, an increasing fluorescence signal is measured online which enables direct assessment of results after PCR without additional detection steps. The test is completed within about 5 h. Two sets of primers and probes were designed and tested: a genus-specific assay for the detection of Enterococcus spp. based on 23S rRNA sequence and an Escherichia coli-specific assay based on the uidA gene sequence. Specificity of the assays was confirmed by testing strains of target bacteria and potential interfering microorganisms. Application of the tests to 55 natural water samples showed the need of an overnight enrichment step to achieve compliance with detection limits of existing regulations. Compared with a parallel microbiological examination of the samples, agreement was 96% with the Enterococcus assay and 98% with the E. coli assay. The rapidity and feasibility of the method point to benefits in drinking water analysis, particularly in emergency situations and, thus, to improved public health management.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Aeromicrobiological studies in the Moscow cathedrals

A microbiological examination of the air has been carried out inside the Moscow Kremlin Cathedrals. Comparison studies on concentrations of airborne microorganisms were performed in different indoor environments -- with and without air-conditioning system, with many and without visitors. The highest values were found indoors with great public attendance and where no air-conditioning system was available. The Gram-positive bacteria were predominant in the air whereas the Gram-negative ones mainly were found on the surface of walls and of stone objects. The majority of airborne microorganisms were capable of producing acid.     

Bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in clinical blood cultures

A bioluminescent assay of bacterial ATP for rapid detection of bacterial growth in 512 clinical aerobic blood cultures was evaluated. At the detection limit of bacterial ATP (10^?10 mol/l) in the blood cultures 94.2% of the true positive blood cultures were detected (sensitivity) and the specificity was 85.8%. If the cut-off limit was increased the sensitivity decreased and the specificity increased and at 2 × 10^?9 mol/l ATP the maximum correctly classified blood cultures was reached. At this cut-off limit the sensitivity was 82.9% and the specificity was 99.6%. In 54.3% of the true positive blood cultures bacterial growth was detected more rapidly with the bioluminescent assay than with macroscopic examination and subculture.     

Use of the bioluminescent method for the determination of bacterial adenosinetriphosphate (ATP-metry) in microbiology

The attention of a wide circle of specialists has recently been attracted by different methods for rapid determination of pathogenic microorganisms in biological specimens, environmental objects and foodstuffs, as well as in cases of possible acts of bioterrorism. In this respect the bioluminescent method for determination of adenosine triphosphate (ATP) contained in microbial cells is of interest. The method is based on the interaction ATP, luciferase and luciferin, accompanied by giving off energy in the form of light emission. When compared with routine methods, the use of this method considerably reduces the duration of the analysis, and its high sensitivity is comparable with that of the polymerase chain reaction. In this review the data on the prospects of the practical use of the bioluminescent method of ATP-metry are presented.     

Measurement of the adenosine triphosphate content of cysts of Globodera rostochiensis as a method for assessing the efficacy of fumigant nematicides

The measurement of the adenosine triphosphate (ATP) content of samples of 50 cysts of Globodera rostochiensis collected from 40 fields in the Netherlands has been tested as a method for determining the mortality of the nematodes achieved by fumigation of these field soils. The technique used is based on bioluminescent photometry and it has been fully described previously. The ATP assessment has been compared with results obtained by the advisory service in the Netherlands using hatching tests based on samples of 100 cysts from the same field soils. The ratio of the values before and after soil treatment showed that the methods usually provided similar estimates of mortality. The relationship between ATP and hatched juveniles was also similar from pre-treatment and post-treatment samples. The amount of ATP, calculated from these results, which is equivalent to the maximum post-treatment hatch allowed for a successful treatment in the Netherlands was used in conjunction with the mortality estimates to assess the success of the fumigation. On this basis the two methods disagreed in the overall assessment of fumigation in only five of the 40 fields. This frequency of disagreement is not significantly greater than expected from the variability of either method. The ATP technique clearly offers a reliable and relatively inexpensive method for estimating the efficacy of fumigant nematicides and is much more rapid than the alternative hatching tests or egg counts. Therefore, it could replace these in situations where the grower wanted a rapid assessment on the success, or otherwise, of his fumigation.     

The use of theVibrio harveyi luminescence mutagenicity assay as a rapid test for preliminary assessment of mutagenic pollution of marine sediments

Mutagenic pollution of the natural environment is currently one of the most serious environmental problems. It includes the pollution of marine sediments. Therefore, rapid detection of the presence of mutagens is an important issue. Recently, we have developed a novel microbiological assay for rapid assessment of mutagenicity of samples from the natural environment. This assay is based on bioluminescence of a mutant Vibrio harveyi strain, and was shown to be useful in testing samples of marine water and plant tissues. Here we demonstrate the usefulness of this assay in preliminary assessment of mutagenic pollution of marine sediments. Mutagenicity of environmental samples taken from the Baltic Sea, is documented and compared here with a commercially available standard sediment sample (IAEA 383), which contains known amounts of mutagenic compounds. The whole procedure, from obtaining a sample in the laboratory to getting final results, is very short (less than 4 h).     

An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA)

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.     

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Measurement of adenine nucleotides in plasma

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A ‘stop solution’ containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg⁺² and Ca⁺² that are co‐factors for many ATPases. Stop solution and blood were mixed using a two‐syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75–7.95) and Mg²⁺ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0–15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright © 2003 John Wiley & Sons, Ltd.     

云冈石窟石质文物表面及周边岩石样品中微生物群落分析

【目的】通过对云冈石窟石质文物表面及云冈石窟周边岩石样品中微生物的研究,建立可用于快速检测石质文物中微生物的方法。【方法】选取云冈石窟石质样品和云冈石窟周边岩石样品作为研究对象,应用PCR-DGGE技术对样品中的微生物群落结构进行了分析研究。【结果】根据系统发育树聚类分析,可以得出云冈石窟中检测出的微生物主要分为四大类群:γ-变形菌纲、鞘脂杆菌门、α-变形菌纲和放线菌纲;根据GenBank数据库中的序列比对结果,可以知道云冈石窟周边类似岩石样品中的微生物主要属于γ-变形菌纲、厚壁菌门和α-变形菌纲等。【结论】本实验成功检测出云冈石窟石质样品表面及云冈石窟周边岩石样品中的微生物类群,为云冈石窟的保护工作提供了有力依据,同时也证明了DEEG和分子克隆技术相结合的方法是检测石质文物中微生物群落结构的一种可操作性强、快速、准确的检测手段。     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。