Rapid determination of microbial contamination of Bupleurum chinense DC. decoction slices by ATP bioluminescence technology combined with statistical analysis methods / 药学学报

This study focuses on the microbial quality control of the Chinese herbal decoction pieces. In view of the shortcomings of traditional culture methods such as slow detection speed and inability to detect unculturable microorganisms, a new method based on ATP bioluminescence technology combined with statistical analysis methods was established to rapidly predict and quantitatively detect the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) contaminated Bupleurum chinense DC. decoction pieces. Based on the optimized ATP bioluminesence detection system, accurate detection of pure bacterial solution of Escherichia coli, Bacillus subtilis and Staphylococcus aureus can be achieved, with detection limits of 47.86, 89.13 and 1 862.09 CFU·mL-1, respectively. The detection time was 6.5 h, and the detection cost was as low as 2 yuan/time. The upper and lower warning limits of TAMC were determined by the misjudgment rates of 10% and 20%, respectively. And the warning limit of TYMC was determined by the misjudgment rate of 20%. The proposed crossing method could quickly predict the amount of microbial contamination in Bupleurum chinense DC. decoction pieces. The constructed partial least squares regression (PLSR) model could accurately quantify the quantity of microbial contamination in Bupleurum chinense DC. decoction pieces. The optimal PLSR prediction model for TAMC had a correction coefficient (R2) of 0.826, a root mean square error of correction set (RMSEE) of 0.468 and a root mean square error of cross-validation set (RMSECV) of 0.465. The R2, RMSEE and RMSECV in the prediction model of TYMC were 0.778, 0.543 and 0.541, respectively. The aim of this study is to establish a kind of rapid detection method and prediction models for the microbial limit of traditional Chinese medicine and Chinese herbal decoction pieces, and to provide a more convenient and sensitive detection technology for the microbial quality process control of traditional Chinese medicine products.

Evaluation on surface cleanliness of personal digital assistant as well as cleaning and disinfection efficacy / 中国感染控制杂志

Objective To investigate the cleanliness status of personal digital assistant(PDA)surfaces, and observe the effect of disposable antiseptic wipes on the cleaning and disinfection of PDA surface.Methods From January to March 2016, 83 daily used handheld PDA in 50 clinical wards of a hospital were as control group, 83 PDA disinfected by disposable antiseptic wipes(CaviWipes)and used in the same environment of the ward at the same time were selected as the intervention group, cleanliness status of PDA surfaces of two groups were detected by adenosine triphosphate(ATP)bioluminescence assay.Results Taken a cut-off value≤100 RLU/100 cm2 as qualified standard of cleanliness, none of PDA in control group was qualified with ATP bioluminescence reading ranged from 189 to 3 379 RLU;after disinfection with CaviWipes, the qualified rate of intervention group was 90.4％(n=75), ATP detection value of PDA surface in all departments were significantly lower than control group(median:[18-28] RLU vs[290-339]RLU, all P＜0.05).Conclusion Microbial contamination on PDA used in hospital wards is common, antiseptic wipes containing quaternary ammonium salt can be used for cleaning and disinfecting surface of PDA.

A comparative analysis of micro-organism colonization of tooth-brushes in a public bath based on the storage technique / 대한구강보건학회지

OBJECTIVES: Bacterial colonization on toothbrushes can cause stomatitis and other oral diseases such as dental caries and periodontitis. In this study we tested 18 toothbrushes, of which six were left exposedto the air, six were stored in a plastic bag, and six in a toothbrush case. METHODS: ATP (RLU: relatively light unit) values measured by ATP Luminometer were compared with APC (CFU: colony forming unit) by swabbing culture method using 3M™ petrifilm™ aerobic count plates. RESULTS: Swabs from toothbrushes left exposed in the air developed an average of 651.67±261.94 CFU, while the “In plastic bag” and the “In toothbrush case” specimens developed 593.17±354.33 and 120.83±68.87 CFU, respectively. CONCLUSION: The best option is to rinse the toothbrush well with water and to keep them dry in a well-ventilated area in order to reduce the growth of bacteria in a public bath.

Cross-sectional survey and analysis of cleaning quality of dental handpieces in Suzhou City / 中国感染控制杂志

Objective To explore the cleaning status and cleaning quality of dental handpieces in various types of medical institutions in Suzhou City.Methods On October 26-31, 2015, dental clinics in the whole city were sampled according to cross-sectional survey and proportional sampling method, the cleaning quality of dental handpieces in each clinic was detected by ATP bioluminescence assay.Results 72 medical institutions, 201 handpieces, 402 samples in 10 administrative regions of the city were sampled, 42 samples was unqualified, unqualified rate was 10.45％, unqualified rate of cleaning of dental handpiece surface was higher than waterline of dental handpiece(17.91％ vs 2.99％, P<0.05).Cleaning quality of dental handpieces in different grades of medical institutions was different(P<0.05), tertiary medical institutions were all ualified, medical institutions without grade was 14.45％.According to the classification based on name of different medical institutions, cleaning quality of handpieces was statistically significant(P<0.05), cleaning efficacy of dental handpieces in department of stomatology of public hospitals was best(unqualified rate was 4.31％), while private dental clinics had the worst cleaning efficacy(unqualified rate was 13.81％).Conclusion Education and training of dental handpieces cleaning in the whole city should be strengthened, especially the management of cleaning of dental handpieces in low grade and private dental clinics.

Study on feasibility of ATP bioluminescence technology applied to disinfection quality monitoring of flexible endoscope / 医疗卫生装备

Objective To explore the feasibility of applying ATP bioluminescence technology to disinfection quality monitoring of the flexible endoscope.Methods Totally 30 flexible endoscopes used repeatedly from October 2014 to March 2015 were randomly selected,and had the disinfection quality monitored by ATP bioluminescence technology and bacterial culture method respectively.Parallel comparison was carried out to evaluate the feasibility of applying ATP bioluminescence technology to disinfection quality monitoring of the flexible endoscope.Results ATP bioluminescence technology showed that the qualification rate of the disinfection was 93.3％,and bacterial culture method found it was 96.7％.The two methods proved the outer surface of the endoscope had the disinfection acceptable while the biopsy hole and intracavity not.There was no significant correlation between the two methods while high consistency between the detection results by the two methods.Conclusion ATP bioluminescence technology can be used for preliminary screening in field,instant and daily monitoring of the medical flexible endoscope,which assists bacterial culture method in disinfection quality monitoring of the flexible endoscope.

Avaliação das boas práticas em uma unidade de alimentação e nutrição escolar de Minas Gerais / Evaluation of good practices in a power unit and school nutrition of Minas Gerais

A mudança no estilo de vida faz com que o consumidor busque cada vez mais alimentar-se fora de casa. Esta mudança exigiu que as Unidades de Alimentação e Nutrição se adequassem às novas legislações para atender à segurança dos alimentos e às exigências dos consumidores. O objetivo desta pesquisa foi investigar e avaliar as condições higienicossanitárias de todo processo de manipulação de alimentos em uma Unidade de Alimentação e Nutrição escolar de Minas Gerais, por meio da aplicação de checklist e técnica de sedimentação em placas e ATP-Bioluminescência. Para avaliar as BPF, foi utilizado o guia de verificação baseado na RDC 216 (2004) e RDC 275 (2002) com 107 itens divididos em blocos, também foi aplicado um questionário para caracterização do perfil dos funcionários da UAN. Para as análises de sedimentação em placa e ATP-Bioluminescência os pontos de amostragem foram escolhidos conhecendo-se o fluxo de produção da UAN, sendo selecionados 9 pontos e 7 funcionários. Observou-se que 46% tinham idade superior a 20 anos e a renda mensal de 77% dos funcionários é de 1 a 2 salários mínimos. Os resultados globais da aplicação da lista de verificação mostraram que a UAN analisada apresentou valores médios de 50% de atendimento nos quesitos avaliados sendo classificada no grupo 3. Para análise de ATP-biolumescência realizadas na área de preparo (PRE), mesa 1 de preparo (ME 1) e mesa 2 de preparo (ME 2), 100% das amostras obtiveram valores considerados inaceitáveis, quando comparados com a recomendação da American Public Health Association (APHA). Pode-se concluir que a UAN avaliada apresentou elevado grau de não conformidades, não atendendo 47 dos 107 itens avaliados necessários ao atendimento das condições higienicossanitárias satisfatórias, estando, portanto, em desacordo com o programa de Boas Práticas de Fabricação.

Application of ATP bioluminescence assay in survey of cleanliness of hos-pital environmental object surfaces and health care workers’hands / 中国感染控制杂志

Objective To investigate cleanliness of hospital environmental object surfaces and hands of health care workers(HCWs).Methods The adenosine triphosphate (ATP)bioluminescence assay was used to detect object surfaces and hands of HCWs in a hospital,on-the-spot intervention was conducted.Results The qualified rates of hospital environmental object surfaces and ventilator-relevant object surfaces were 58.14% (200/344)and 69.88%(116/166)respectively,the qualified rate of ventilator tracheal intubation site was low (29.17%);the qualified rate of telephone surfaces was the lowest (27.27%).The qualified rates of ventilator-relevant object surfaces used con-tinuously for ≥48h and <48 h were 56.70%(55/97)and 88.41 %(61/69)respectively,there was significant differ-ence between the two(χ2 =19.26,P <0.01).The qualified rates of HCWs’hands before and after intervention were 34.18% and 85.58% respectively,relative light unit (RLU)values were (1 033.46±106.20)and (80.46±10.68) respectively,the qualified rates and RLU before and after intervention were both significantly different (both P <0.01).Conclusion Contamination of object surfaces and hands’of HCWs in hospital dynamic environment is seri-ous,ATP bioluminescence detection and on-the-spot intervention is helpful for improving cleanliness of hospital en-vironment object surfaces and HCWs’compliance to hand hygiene.

Application of ATP bioluminescence assay in evaluating cleaning quality of medical devices / 中国感染控制杂志

Objective To explore the application of adenosine triphosphate (ATP )bioluminescence assay in evaluating cleaning quality of medical devices.Methods Manual and machine cleaned medical devices were randomly selected from central sterile supply department of a hospital in 2011 -2013,cleaning quality was detected with ATP biolumi-nescence assay,relative light unit (RLU)value was determined to assess the cleaning quality.Results A total of 460 pieces of cleaned devices were detected in 2011 -2013,404 (87.83%)were qualified.The qualified rates of manual cleaning and machine cleaning were 70.73% and 94.07% respectively,the qualified rates of general surgical devices and lumen devices were 90.71 % and 81 .76% respectively,there were significant differences in qualified rates of different cleaning methods and different categories of medical devices (both P <0.01 ).The qualified rates of manual cleaning in 2011 - 2013 were 32.35%,79.63%,and 94.29% respectively,machine cleaning were 79.45%,98.15%,and 98.08% respectively;differences in qualified rates of manual cleaning and machine cleaning in different years were significant (all P <0.01 ).Conclusion ATP bioluminescence assay can be used for evalua-ting cleaning quality of medical devices.

Application of ATP bioluminescence assay in surveillance of terminal disinfection of effects / 国际检验医学杂志

Objective To summarize the application of ATP bioluminescence assay in surveillance of terminal disinfection of effects ,so as to provide the basis for intervention of disinfected effects .Methods ATP bioluminescence assay were employed to randomly test the surfaces of operating objects in therapeutic rooms and beside tables in wards ,total 144 object surfaces ,of each clinical departments in the whole hospital .The values of ATP bioluminescence assay were read on‐site ,0-250 RLU was recognized as qualification ,while disqualification when >250 RLU .The disqualified object surfaces were performed on‐site intervention that all of them were re‐disinfected ,the results were compared .Results Both the surfaces of operating objects and beside tables were dis‐qualified before disinfection ,and the values of ATP bioluminescence assay were 780 ± 10 .34 RL and 853 ± 13 .29 RLU respectively . The pass rates of ATP bioluminescence assay was 61 .97% of operating surfaces and 79 .45% of beside table surfaces the first dis‐infection .The disqualified sites were retested following on‐site intervention .The values of ATP bioluminescence assay were 431 .02 ± 0 .53 before intervention and 1 .43 ± 0 .59 after intervention ,and the difference was statistically significant .Conclusion ATP bi‐oluminescence assay can get more immediately ,simple and timesaving in evaluating the effect of disinfection and estimate the effi‐ciency of disinfection timely ,which can also provide the scientific basis on on‐site intervention so as to improve the execution power of hospital infection management .

Disinfection efficiency of N-doped nano-TiO2 on the curing light-guide rods / 实用口腔医学杂志

Objective:To observe the disinfection efficiency of N-doped nano-TiO2 on the curing light-guide rods.Methods:50 light-guide rods from 6 dental clinics were included,the bacteria adhesion on the light-guide rods before and after disinfection with N-doped nano-TiO2 was investigated using ATP bioluminescence method.Results:The RLU value of light-guide rods exposed to the air was 51 -649 before the use of N-doped nano-TiO2 disinfection,after spraying N-doped nano-TiO2 ,the RLU value of light-guide rods was 0 -6(P <0.01 ).Conclusion:N-doped nano-TiO2 has significant sterilization effect on the curing light-guide rods.

ATP-Bioluminescence as a method to evaluated microbiological quality of UHT milk / ATP-Bioluminescência como método para avaliação da qualidade microbiológica de leite UAT

New approaches are needed to quickly indicate possible contamination of UHT milk, among them the technique of ATP-Bioluminescence. Therefore, the aim of this study was to compare the results of culture methods with the results of ATP-Bioluminescence technique of 102 UHT whole milk samples incubated at 48, 72, and 168 hours. UHT milk samples were analyzed for the presence of mesophilic and psychrotrophic aerobic microorganisms using Plate Count Agar (PCA), Brain-Heart Infusion (BHI) media and PetrifilmTM Aerobic Count (AC) plates. The ATP-Bioluminescence technique was applied through the Microbial Luminescent Screening (MLS) system. Significant correlations were found between counts of aerobic mesophilic microorganisms on PCA, PetrifilmTM AC, BHI and results of ATP bioluminescence technique (P≤0.05). The ATP-Bioluminescence technique had higher correlation with counting method in PCA than BHI media. At lower pass/fail limits of Relative Light Units (60, 50, 45 and 40 RLU), the number of samples identified as positive increased and statistically agreed with aerobic mesophilic microorganism counts (P>0.05). For the dairy industry, the ATP-Bioluminescence technique may become an important tool that assists the official methods to quickly monitor the microbiological quality of UHT milk though this will likely require a threshold below 150 RLU...

Novos métodos são necessários para detectar de forma rápida a contaminação do leite UAT, entre eles, a técnica de ATP-Bioluminescência. Portanto, objetivou-se comparar os resultados de métodos de cultura tradicionais com os resultados de ATP-Bioluminescência de 102 amostras de leite UAT integral incubadas por 48, 72 e 168 horas. Os leites UAT foram analisados quanto à presença de micro-organismos aeróbicos mesófilos e psicrotróficos usando os ágares PCA, BHI e placas PetrifilmTM AC. A técnica de ATP-Bioluminescência foi aplicada por meio do sistema Microbial Luminescent Screening (MLS). Significantes correlações foram obtidas entre as contagens de micro-organismos mesófilos aeróbios em PCA, PetrifilmTM AC, BHI e os resultados da técnica de ATP-Bioluminescência (p<0,05). A técnica de ATP-Bioluminescência tem maior correlação com o método de contagem em meio PCA que BHI. Quando valores limites de Unidades Relativas de Luz (60, 50, 45 e 40 RLU) foram menores, o número de amostras identificadas como positivas aumentou e concordou estatisticamente com a contagem de micro-organismos mesófilos aeróbios (P>0,05). Para as indústrias de laticínios, a técnica de ATP-Bioluminescência pode se tornar uma ferramenta auxiliar aos métodos oficiais para o monitoramento rápido da qualidade microbiológica do leite UAT, desde que sejam utilizados limites abaixo de 150 RLU...

Avaliação da qualidade microbiológica de bebida láctea e creme de leite UAT por ATP-Bioluminescência / Evaluation of microbiological quality of UHT milk drink and UHT milk cream by ATP-Bioluminescence

Embora métodos tradicionais sejam utilizados na avaliação microbiológica de produtos UAT, metodologias rápidas, baseadas em ATP-Bioluminescência, têm sido desenvolvidas. Os resultados da aplicação dessa técnica em 54 amostras de bebida láctea UAT achocolatada e 12 de creme de leite UAT foram comparados com os resultados de métodos microbiológicos, utilizando-se diferentes meios de cultura e tempos de incubação das referidas amostras. A técnica de ATP-Bioluminescência foi aplicada por meio do sistema MLS, e os resultados foram expressos em unidades relativas de luz (RLU). Em todos os tempos de incubação - 48, 72 e 168 horas - , as amostras apresentaram contagens baixas de microrganismos mesófilos e psicrotróficos aeróbios quando analisadas em meio PCA, BHI, PetrifilmTM AC e por ATP-Bioluminescência (<150 RLU), demonstrando alta especificidade da técnica. Apenas uma amostra de creme de leite UAT apresentou contagem de mesófilos aeróbios acima do padrão estabelecido pela legislação brasileira (<100 UFC/mL) quando analisada em meio PCA (260 UFC/mL) e PetrifilmTM AC (108 UFC/mL), no tempo de 168 horas. Essa alta contagem de microrganismos mesófilos aeróbios também foi detectada pela técnica de ATP-Bioluminescência (416 RLU). Os resultados da técnica de ATP-Bioluminescência foram iguais aos resultados em meio PCA, BHI e PetrifilmTM AC.

Although traditional methods are used for the microbiological evaluation of UHT products, rapid methodologies based on ATP-Bioluminescence have been developed. The results of applying this technique in 54 samples of chocolate UHT milk drink and 12 of UHT milk cream were compared with the results of microbiological methods, using different culture media and incubation times for the referred samples. The ATP-Bioluminescence technique was applied through the MLS system and the results were expressed as relative light units (RLU). In all incubation times - 48, 72, and 168 hours - , the samples showed lower counts of mesophilic and psychrotrophic aerobic microorganisms when analyzed using PCA, BHI, PetrifilmTM AC and ATP-Bioluminescence (<150RLU), demonstrating the technique's high specificity. Only one sample of UHT milk cream showed a mesophilic aerobic count above the standard established by Brazilian legislation (<100CFU/mL) when analyzed in PCA (260 CFU/mL) and PetrifilmTM AC (108CFU/mL) at 168 hours. This high count of aerobic mesophilic microorganisms was also detected by the ATP-Bioluminescence (416 RLU) technique. The results of the ATP-Bioluminescence technique were equal to the results in PCA, BHI and PetrifilmTM AC.

Avaliação do treinamento de manipuladores de alimentos de restaurantes comerciais pelo ensaio ATP-bioluminescência / Evaluation of food handlers training in commercial restaurants by using ATP bioluminescence methodology

Os programas de treinamentos para manipuladores de alimentos são meios eficazes para transmitir conhecimentos e promover melhorias nas condições higiênico-sanitárias em estabelecimentos alimentícios.Neste trabalho foi avaliado o efeito do treinamento em boas práticas de manipulação de alimentos,ministrado aos funcionários de restaurantes comerciais da região de Ouro Preto – MG, por meio do teste de ATP-bioluminescência. O teste foi utilizado para quantificar a carga orgânica, medida como valor de“Unidades Relativas de Luz – URL”, nas amostras coletadas das mãos de manipuladores, de bancadas de trabalho e de superfícies de utensílios, selecionadas aleatoriamente em cada restaurante. Após o treinamento,houve melhoria no padrão higiênico, evidenciado pela redução da carga orgânica nas superfícies avaliada sem todos os restaurantes. Apesar da redução dos valores de URL, algumas superfícies não alcançaram o valor recomendado para superfície em condições higiênicas satisfatórias. A aplicação do teste ATPbioluminescência para avaliação do treinamento de manipuladores mostrou ser eficaz e pode ser empregado em associação com os tradicionais métodos de contagem de micro-organismos em treinamentos realizado sem Unidades de Alimentação e Nutrição. Apesar da redução dos valores de URL indicar a melhoria nas condições higiênicas, o trabalho de treinamento e monitoramento deve ser continuado.

Training programs designed for food handlers are an effective way to spread the knowledge and topromote improvements on the hygienic conditions of food establishments. This study aimed at evaluatingthe effect of a training course on good handling practices for food handlers of selected restaurants in theregion of Ouro Preto, MG, Brazil, by using ATP-bioluminescence assay. This test was used to quantifythe organic load, measured by “Relative Light Units – RLU”, in samples collected from workers hands,surfaces of work benches and utensils, which were randomly selected in each restaurant. After training,a significant improvement in the hygienic conditions was observed, as evidenced by the decrease in theRLU values in samples from all restaurants. Despite the decreased RLU values, some surfaces did notreach the recommended value for surfaces samples in the satisfactory hygienic conditions. The ATPbioluminescencetest showed to be effective for evaluating the training success, and could be a powerfultool when applied in training programs at food services, and in association with the conventionalmicrobiological methodologies. In spite of the reduction in RLU values, indicating.

Clinical research on chemotherapy of recurrent and refractory non-Hodgkin lymphoma directed by ATP bioluminescence chemosensitivity assay in vitro / 白血病·淋巴瘤

ObjectiveTo investigate the clinical value of ATP bioluminescence tumor chemosensitivity assay (ATP-TCA) for recurrent and refractory non-Hodgkin lymphoma (NHL) specimens in vitro.Methods Thirty-four freshlytaken recurrent andrefractoryNHL specimens weretestedin vitro for cancer chemosensitivity by ATP-TCA.ResultsDrug sensitivity of NHL specimens had heterogeneity.Different drugs had different tumor growth inhibition ratio in vitro.Response rate (RR) of the patients receiving chemotherapy according to in vitro assay was 82.4 ％ (28/34),complete response rate (CR) was 52.9 ％ (18/34).In DICE group RR was 60.0 ％ (18/30),CR rate was 33.3 ％ (10/30).In GDP group RR was 62.3 ％ (33/53),CR rate was 26.4 ％ (14/53).In ATP-TCA group RR was significantly higher than those in DICE and GDP groups (x2 =3.93,P =0.047; x2 =3.98,P =0.046).ConclusionThe results of ATP-TCA assay are correlated well with clinical treatment responses.The assay may be an important and useful method for individual-based chemotherapy of cancers.

Evaluation of Hygienic Status using ATP Bioluminescence Assay and Food Service Workers' Sanitation Performance in Elderly Welfare Facilities

The purpose of this study was to investigate food sanitation status in elderly welfare facilities and assess the performance of food sanitation practices. Twenty elderly welfare facilities out of 85 located in Seoul with a capacity of fewer than 50 persons participated. The food sanitation status of worktable, kitchen utensils (knives, cutting boards, ladles, spoons), and tableware and bowls were examined by ATP bioluminescence. The results found that the ATP value of knife was the highest. Those of ladles appeared relatively higher than others. Meanwhile, the tableware and bowls, although washed everyday after meals, had the lowest ATP value. This study also conducted a survey on the food sanitation practices of 32 cooking employees in the 20 facilities. Fifty-six percent were in their 40s, and 53% had graduated from high school. More than half (66%) of them had no certification of cooking. Half of the respondents had worked for at least 5 years in food service facilities, and had received food sanitation training. Among them, 31% said they applied food sanitation training while working, and 47% responded the training was very helpful. The foodservice employees demonstrated good food sanitation practices. The results show that food sanitation performance of the workers significantly differed according to their age, education level, total work experience in food service facilities, chef certification, and prior food sanitation experience.

Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection / 药物分析学报

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.

Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection / 西安交通大学学报·英文版

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase, equipped with a polyhistidine affinity tag, was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995 mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108, and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein, the ATP content of bacteria was 9.48×10-16mol/mL, and was identical to the bacteria counts (4 500 CFU/mL) in order of magnitude. Taken together, our results provided a simple and efficacious method of the preparation of recombinant luciferase, which could be applied in the determination of bacteria via ATP bioluminescence.

Interference of some organic substances and microorganisms adhered to stainless steel in ATP-bioluminescence measurement

Adhesion of organic substances and microorganisms on stainless steel surface was evaluated. The log RLU measurement was affected at a lower or higher degree, by type or concentration of the organic substances (casein, lipid, sucrose and their combinations) adhered to surface. However, if the samples analyzed were from the food processing surfaces, they would have been considered to be under satisfactory hygienic conditions with log RLU < 2.18 (< 150 RLU). The log RLU measurements of Staphylococcus carnosus-adhered surfaces, combined with or without the organic substances, were higher (p<0.05) than those of the Bacillus subtilis spores-adhered surface, regardless of the concentration. The samples taken from the surfaces adhered with the casein, lipid, sucrose and 2.9x10¹ DMC.cm-2 and 2.9x10² DMC.ml-1 did not have any significant differences (p>0.05) on ATP-bioluminescence measurements. However, the surfaces adhered with 2.9x10³ DMC.cm-2 of B. subtilis spores and organic substances showed an increase in the log RLU measurement compared to surfaces containing only the organic substances.

A adesão de substâncias orgânicas e de microrganismos em cupons de aço inoxidável ASI 304, #4 foi avaliada. A medida de Unidades Relativas de Luz (RLU) foi afetada em menor ou maior nível pelo tipo e concentração das substâncias orgânicas e suas combinações. Entretanto, se as amostras fossem oriundas de superfícies de processamento de alimentos, elas seriam consideradas em boas condições higiênicas pois as medidas de log URL ficaram abaixo de 2,18 log (< 150 URL) com ou sem adição das substâncias orgânicas e independente da concentração estudada, as células vegetativas apresentaram número de log URL para S. carnosus maior (p<0,05) do que para os esporos de B. subtilis. Amostras de superfícies aderidas com caseína, lipídio e sacarose e 2.9x10¹ CDM.cm-2 ou 2.9x10² CDM.ml-1 não apresentaram diferença significativa (p>0.05) na medida de ATP-bioluminescência. Entretanto, superfícies aderidas com 2.9x10³ CDM.cm-2 de esporos de B. subtilis e as substâncias orgânicas mostraram um aumento no log URL comparado com as superfícies contendo somente substâncias orgânicas.