可视化和实时荧光环介导恒温扩增技术快速检测原料乳中的沙门氏菌

【背景】原料乳质量控制是乳制品生产流程中的关键环节,沙门氏菌是污染原料乳的主要致病菌之一。随着生物检测技术的不断发展,建立用于快速检测原料乳中沙门氏菌的实用型方法具有重要意义。【目的】评价可视化环介导恒温扩增技术(visual loop-mediated isothermal amplification,V-LAMP)和实时荧光环介导恒温扩增技术(real-time fluorescence loop-mediated isothermal amplification,RF-LAMP)方法快速检测原料乳中沙门氏菌的实效性。【方法】建立沙门氏菌LAMP反应体系和反应条件,对比V-LAMP和RF-LAMP两种方法的特异性、灵敏度、稳定性及其应用于实际样品检测的效用。【结果】采用V-LAMP和RF-LAMP方法检测沙门氏菌标准菌株,其结果均为阳性,检测非沙门氏菌标准菌株其结果均为阴性;该两种方法的检出限为13 CFU/mL,检测重复率均为100%;经检测10份人工污染样品,该两种方法的检测结果与实际结果高度一致;经检测73份实际样品,相对于标准方法,该两种方法其相对特异性分别为94.12%和92.65%,相对敏感度分别为100%和100%,相对准确率分别为94.52%和93.15%,一致性χ2值分别为2.25和3.20,与标准方法无明显差异(P>0.05)。【结论】V-LAMP和RF-LAMP方法检测特异性好、灵敏度高、稳定性好,适用于原料乳中沙门氏菌的快速检测。     

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods

Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.     

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin‐producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time‐consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non‐O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti‐O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time‐resolved fluorescence immunoassay (TRFIA). Copyright © 2002 John Wiley & Sons, Ltd.     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

An automated fluorescence videomicroscopy assay for the detection of mitotic catastrophe

Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and – in this context – revealed an important role of p53 in the control of centrosome number.     

Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses

Aims: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. Methods and Results: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi‐hard uncooked cheese and ‘Camembert’ type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim–tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1–10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5·10⁴ CFU ml^?1 after 24‐h incubation at 41·5°C in this medium. Conclusions: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. Significance and Impact of the Study: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.     

A comparative study of the Gene-Trak Listeria assay, the Listeria-Tek ELISA test and the FDA method for the detection of Listeria species in raw milk

A total of 110 raw milk samples were analysed using the Gene-Trak Listeria assay, unmodified and with four modifications to its enrichment procedure, the Listeria-Tek ELISA test and the FDA method. Thirty-five samples yielded confirmed positive results by at least one of the methods tested. The original Gene-Trak protocol yielded 24 confirmed positive results, significantly higher than the 13 obtained with the Listeria-Tek ELISA test or with the FDA method. The modifications to the Gene-Trak Listeria assay did not significantly increase the detection rate of Listeria spp.     

Sensitive high-performance liquid chromatographic assay for norfloxacin utilizing fluorescence detection

A rapid, sensitive and reproducible reversed-phase high-performance liquid chromatographic assay was developed for the determination of norfloxacin. Following protein precipitation with 10% trichloroacetic acid, norfloxacin and the internal standard enoxacin were extracted from plasma with chloroform, dried and reconstituted in the mobile phase. The chromatographic separation of norfloxacin and the internal standard enoxacin was achieved on a C₈ column with fluorescence detection set at 280 and 418 nm for excitation and emission, respectively. The peaks with a resolution factor greater than 1.5 were free from interferences. Excellent linearity (r² 0.998) was observed over the concentration range 0.025–5.0 μg/ml in plasma. The inter-assay variability was 13.6% or less at all concentrations examined. The suitability of the assay for pharmacokinetic studies was determined by measuring norfloxacin concentration in rat plasma after administration of a single intravenous 10 mg/kg dose.     

Development of a nanoparticle-based surface-modified fluorescence assay for the detection of prion proteins

A nanoparticle-based immunoassay for the detection of recombinant bovine prion protein (PrP) was developed as a step in the development of screening tools for the prevention of the spread of transmissible spongiform encephalopathies. The assay is based on the competitive binding between PrP and a peptide-fluorophore to a nanoparticle-labeled antibody which is specific for a conserved prion sequence. The fluorophore, when bound to the antibody, is subject to surfaced-modified fluorescence, enabling detection of changes in the concentration of bound fluorophore in the presence of prion protein. Important factors considered during the development of the assay were ease of use, robustness, and detection level. The effects of pH and nanoparticle conjugation chemistry on surface-modified fluorescence observed in the assay were explored. Effects of concentrations of antibody and fluorophore on reproducibility and detection limits were examined. At present, the detection limits of the system are approximately equal to the antibody-peptide fluorophore equilibrium dissociation constant, which is near one nanomolar concentration. Improved assay performance could be obtained by optimization of the nanoparticle surface resonance effects. The simplicity of the assay and ease of use may make the type of assay described in this report attractive for screening purposes in the food industry.     

Anisakis pegreffii: a quantitative fluorescence PCR assay for detection in situ

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.     

The magnetic immuno-polymerase chain reaction assay for the detection of Campylobacter in milk and poultry

L. DOCHERTY, M.R. ADAMS, P. PATEL AND J. McFADDEN. 1996. A rapid and sensitive technique, based on the magnetic immuno-polymerase chain reaction assay (MIPA), was developed for the detection of Campylobacter jejuni in milk and chicken products. Target bacteria are captured from the food sample by magnetic particles coated with a specific antibody and the bound bacteria then lysed and subjected to PCR. The MIPA could detect 420 cfu g^-1of chicken after 18 h, 42 cfu g^-1after 24 h, and 4.2 cfu g^-1after 36 h enrichment. For artificially contaminated milk 63 cfu ml^-1could be detected after 18 and 24 h and 6.3 cfu ml^-1after 36 h enrichment.     

Universal SNP genotyping assay with fluorescence polarization detection

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.     

Development of a fluorescence assay for the detection of L-ficolin-MASP in serum or purified samples

A fluorescence assay for the detection of L-ficolin-MASP in human serum or purified sample was developed by measuring the cleavage of fluorescent amide substrate by L-ficolin associated MASPs bound to the lipoteichoic acid (LTA). LTA (Staphylococcus aureus DSM 20233) was coated on NuncMaxisorp microtiter plates and serum or purified sample incubated overnight at 4 degrees C to allow the L-ficolin-MASP to bind LTA. Assay conditions for binding and complete cleavage of fluorescent amide substrate were standardized. The optimum temperature, incubation time and molarity of NaCl for LTA-ficolin binding were found to be 4 degrees C for 6 h at 1 M NaCl concentration. The optimum incubation time and pH for complete cleavage of fluorescent amide substrate by LTA bound L-ficolin associated MASP were found to be 2 h at pH 8.5. LTA-ficolin binding was found to be highly specific and was inhibited completely by LTA but not with mannose. A calibration curve was prepared by using the purified ficolin-MASP complex (1 to 12 mug/ml) and could be used to find concentration of ficolin-MASP complex in normal human serum.     

Quantum dot-based fluorescence immunosorbent assay for the rapid detection of bacitracin zinc in feed samples

Bacitracin zinc (BAC), a polypeptide antibiotic, is utilized as a feed additive due to its ability to promote growth in animals. However, the abuse of BAC can lead to a great threat to food safety. Therefore, there is an urgent need to develop a rapid and sensitive detection method. In this study, a monoclonal antibody (mAb) against BAC with excellent sensitivity and specificity was obtained. For the first time, quantum dots (QDs) were conjugated with the prepared mAb against BAC and rabbit anti-mouse antibody to fabricate a direct and an indirect competitive fluorescence-linked immunosorbent assay (dc-FLISA and ic-FLISA) to detect BAC. The IC₅₀ of dc-FLISA and ic-FLISA were 0.28 ng/ml and 0.17 ng/ml, respectively. The limits of detection were 0.0016 ng/ml and 0.001 ng/ml, respectively, and the detection ranges were 0.0016–46.50 ng/ml and 0.001–35.65 ng/ml, respectively. In addition, the recovery rate of the two methods ranged from 93.5% to 112.0%, and the coefficient of variation (CV) was less than 10%. Therefore, the methods developed in this work have the merits of low cost, simple operation, and high sensitivity, which provide an effective analytical tool for BAC residue detection in feed samples.     

PCR-based detection of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making

AIMS: To define PCR-based detectability of Staphylococcus aureus in raw milk and intermediate products of raw milk cheese making in the presence of a complex background microflora by targetting different specific genes harboured by a single strain. METHODS AND RESULTS: The strain Staph. aureus FRI 137 harbouring nuc, sec, seg, seh and sei genes was used in this study. Raw milk artificially contaminated by different concentrations of Staph. aureus FRI 137 was employed in dairy processing resembling traditional raw milk cheese making. Samples of milk and curds were PCR-analysed after DNA extraction by targetting all the above genes. The pathogen was detected when the initial contamination was 10(4) CFU ml(-1) by amplification of nuc and seh genes. 10(5) and 10(7) CFU ml(-1) were needed when seg or sei and sec genes were targetted, respectively. Enrichment cultures from raw milk and curd samples proved to increase the detection limit of 1 log on average. CONCLUSIONS: The direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of Staph. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to the real case in order to determine the level of contamination potentially predictable. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection in real dairy systems of significant loads of Staph. aureus by multiple targets PCR can be more accurate.     

Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions

Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0 M) prior to the luminescent enhancement step. [Nle⁴,d-Phe⁷]-α-melanocyte-stimulating hormone (NDP-α-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries.     

Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef.

A DNA probe endolabeled with digoxigenin by PCR was developed to detect and enumerate enterotoxigenic Clostridium perfringens in raw beef. After 2 h of hybridization, membranes were developed by using an anti-digoxigenin-alkaline phosphatase conjugated antibody. The resulting chromogenic reaction allowed us to detect and enumerate < or = 10 CFU of C. perfringens per g.