酶联免疫吸附试验检测艰难梭菌A毒素

实验用兔单特异抗艰难梭菌Ａ毒素ＩｇＧ包被酶标板,以羊抗艰难梭菌Ａ毒素ＩｇＧ标记辣根过氧化物酶作为第二抗体,采用双抗体夹心ＥＬＩＳＡ法检测艰难梭菌Ａ毒素,可检测出０．９４ｎｇ的精制Ａ毒素,对６１株菌的培养液及６５份健康人粪便标本检测发现此法具有较高的特异性。用平行线定量法对几份典型产毒培养物进行了定量测定,结果表明,在一定剂量范围内线性及平行性好,结果准确、可靠。可用于临床粪便标本中艰难梭菌Ａ毒素的筛查及定量检测。     

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.     

A method of enhancing verocytotoxin production by Escherichia coli

The number of verocytotoxin producing Escherichia coli (VTEC) present in the faeces during an infection may be very low, making their detection difficult. We report a method for enhancing toxin production by VTEC using mitomycin C as an inducing agent with the aim of improving the detection of VTEC. In pure culture, mitomycin C enhanced toxin production up to 100-fold. When applied to mixed faecal culture, toxin could be detected in mitomycin C treated samples when standard cultures were negative and when substantially fewer verocytotoxin-producing bacteria were present. Use of this method may aid in the detection of VTEC and is appropriate for use in the routine diagnostic laboratory.     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.     

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.     

Detection of toxin and evaluation of the degree of toxin-formation of Corynebacterium diphtheriae using immunoenzyme analysis

Toxin production and the intensity of toxin formation in 265 C. diphtheriae strains circulating in different areas of the USSR have been studied by the method of the enzyme-linked immunosorbent assay (ELISA). This study has been carried out with the use of the assay system consisting of monoclonal antibodies to the COOH-area of the B-fragment of the toxin molecule adsorbed onto the surface of polystyrene plates, affinity-purified polyclonal antidiphtheria antibodies labeled with horse-radish peroxidase and substrate indicator mixture (5-aminosalicylic acid and hydrogen peroxide). Some specific features of using ELISA for the detection of C. diphtheriae toxin directly in liquid culture medium are presented. High sensitivity, specificity and good reproducibility of this method permitting the detection of C. diphtheriae toxin and the determination of the intensity of toxin formation in the C. diphtheriae strains under study are shown. The method may be recommended for practical use at health institutions.     

Chromatographic analysis of Fusarium toxins in grain samples

Summary A method for the analysis of zearalenone, T-2 toxin, neosolaniol and HT-2 toxin from the grains of barley, wheat and oats has been developed. Toxins are extracted with ethyl acetate, purified on a kieselgel TLC-plate and analysed on a HPTLC-plate. The limits of detection are 0.2 mg/kg for zearalenone and T-2 toxin and 5 mg/kg for neosolaniol and HT-2 toxin. For more accurate estimation the purified toxins are analysed as their trimethysilyl derivatives by gas chromatography, in which the detection limit for all toxins in 50 g/kg and the accuracy ±10%–30%. The percentage recovery in both methods is 80%.     

HPLC-MS-Method for A-Trichothecenes

Gaschromatography with electron capture detection belongs to the most important methods for analysing the A-trichothecenes T-2 toxin, HT-2 toxin and diacetoxyscirpenol. The need of a derivatisation for these mycotoxins prior detection is the main disadvantage of this method. In this paper a sensitive high performance liquid chromatography method to analyse the A-trichothecenes in grain is described. The clean up was performed with multifunctional columns (MycoSep^TM, Romer Labs, Inc., MO). The detection limits of these methods are 3.5 μg/kg for T-2 toxin, 6.1 μg/kg for HT-2 toxin and 4.1 μg/kg for diacetoxyscirpenol. Limits of quantification were 12.7 μg/kg for T-2, 21.9 μg/kg for HT-2 and 14.8 μg/kg for DAS, respectively recovery rates were about 100% for each compound.     

Detection of toxin translocation into the host cytosol by surface plasmon resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER(6-12). To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)(13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.     

Direct detection and quantification of Alternaria alternata lycopersici toxins using high-performance liquid chromatography-evaporative light-scattering detection

Alternaria alternata lycopersici toxins are produced by the tomato pathogenic fungus A. alternata f. sp. lycopersici. They are primary determinants for stem canker disease of tomato and induce apoptosis in animals and plants. Due to the lack of a UV chromophore, their detection has relied on derivatizations or immunoassays, which require tedious pre-assay processes. Here, we describe a method for direct and quantitative detection of the toxins. The method employs an evaporative light-scattering detector (ELSD) coupled to HPLC. It gives a linear response in the range of 400-4000 ng toxin, with a detection limit of about 6 ng/mul. The detection of all major toxin congeners by HPLC-ESLD was confirmed by liquid chromatography-mass spectrometry. Thus, the method provides a simple and sensitive tool for the studies of this group of host-specific mycotoxins.     

Rapid Detection of Clostridium botulinum Toxin by Capillary Tube Diffusion

A micro capillary agar-gel diffusion system for the detection of botulinal toxin in foods and cultures was developed and evaluated. Toxins types A, B, and E, produced in culture broth with and without added trypsin, and type E toxin, produced in inoculated canned clams, were tested with this system and with the mouse bioassay procedure. With nontrypsinized toxin, the capillary diffusion system detected as little as 100 minimal lethal doses (MLD) per ml but was effective only at higher levels, 10(6) to 1.5 x 10(7) MLD/ml, when used with trypsinized toxin. The inability to detect lower levels of trypsinized toxin was due to thioglycolate present in the medium used to produce toxin. Evidently, trypsinization of toxin produces polypeptides still held together by disulfide bonds. Cleavage of these bonds by reduction with thioglycolate reduces the sensitivity of the capillary method. Trypsinized toxin produced in broth without thioglycolate was detected as readily as nontrypsinized toxin. Toxin was detected in canned clams containing as low as 100 MLD/ml. No cross-reactions were observed with type E toxin and types A and B antitoxins. Extensive studies using the capillary method for detecting types A and B toxins were not performed; however, a suspected sample of commercially canned mushrooms gave a positive type B reaction but not a type A reaction. This typing was confirmed later by the mouse bioassay. Toxin was present at a level of 100 MLD/ml. The procedure developed may prove useful as a rapid screening method for the detection of botulinal toxin in foods, with final identification made by using the mouse bioassay.     

Monoclonal antibodies labeled with colloidal gold for immunochromatographic express analysis of diphtheria toxin

A one-step immunochromatographic method for the detection of the diphtheria toxin in different water samples (phosphate buffer, milk, and human nasopharyngeal swab) was developed using a conjugate of monoclonal antibodies labeled with colloidal gold. The detection limit of diphtheria toxin was 10 ng/ml, the time of the analysis was 15 min. The use of silver to enhance the reaction sensitivity and scanning equipment to evaluate the immunochromatography results decreased the detection limit to 1.25 ng/ml.     

Disposable immunosensor for the determination of domoic acid in shellfish

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.     

兰州地区腹泻患者艰难梭菌的感染状况调查

目的调查兰州地区腹泻患者中艰难梭菌的流行特点,揭示国内艰难梭菌感染的现况。方法通过细胞毒检测试验和酶联免疫吸附试验对206份临床粪便样品进行毒素检测。结果 206份粪便滤液经细胞毒检测有26份样品使非洲绿猴肾细胞（vero细胞）圆缩化,确认含有艰难梭菌毒素;经酶联免疫吸附试验有28份为阳性,其中23份与细胞毒检测结果一致,与细胞毒试验的符合率为88.5%。结论兰州地区住院腹泻患者中艰难梭菌感染率约为12.62%。     

Production and analysis of citrinin in corn.

A convenient method for the production and analysis of citrinin in corn is described. Up to 2.964 g of citrinin can be produced by Penicillium citrinum per kg of corn by harvesting on day 21 or later. The analysis method has a lower detection limit of 0.25 ppm. Heating citrinin-contaminated corn destroys citrinin but may produce another toxin instead.     

An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples

Abstract We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells. The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic. Using the HEp-2 cell assay, we examined the distribution and contamination of B. cereus strains that produced an emetic toxin in various foods. Although there were 228 enterotoxin producers among 310 B. cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin). All of the strains that produced the cytostatic toxin were of the H.1 serotype.     

Type A-trichothecenes — Quantitative analysis using LC-MS and occurrence in Austrian maize and oats

During this study a method was developed for the quantitative determination of diacetoxyscirpenol (DAS), T-2 toxin and HT-2 toxin using deuterated T-2 toxin (T-2 d3 toxin) as an internal standard. The described method involves a clean up step of maize extract by the use of Mycosep® 227 columns a chromatographic separation on a Zorbax® bonus-RP-column (2,1×150mm) and the detection and quantification step on a mass spectrometer in SIM (Selected Ion Monitoring) mode.Data on the occurrence of three type A trichothecenes in Austrian maize, maize silage and oats were collected. It could be shown that maize and silage samples harvested in 2002 were only contaminated to a small extent with T-2 toxin (8% of the maize, 0% of the silage) and with HT-2 toxin (30% of the maize, 18% of the silage). But most of the analysed oats samples showed significant levels of T-2 toxin (64%) or HT-2 toxin (82%).     

Rapid and sensitive detection of microcystin by immunosensor based on nuclear magnetic resonance

A stable and sensitive toxin residues immunosensor based on the relaxation of magnetic nanoparticles was developed. The method was performed in one reaction and offered sensitive, fast detection of target toxin residues in water. The target analyte, microcystin-LR (MC-LR) in Tai lake water, competed with the antigens on the surface of the magnetic nanoparticles and then influenced the formation of aggregates of the magnetic nanoparticles. Accordingly, the magnetic relaxation time of the magnetic nanoparticles was changed under the effect of the target analyte. The calibration curve was deduced at different concentrations of the target analyte. The limit of detection (LOD) of MC-LR was 0.6 ng g⁻¹ and the detection range was 1–18 ng g⁻¹. Another important feature of the developed method was the easy operation: only two steps were needed (1) to mix the magnetic nanoparticle solution with the sample solution and (2) read the results through the instrument. Therefore, the developed method may be a useful tool for toxin residues sensing and may find widespread applications.     

A rapid and sensitive assay for pertussis toxin based on pigment aggregation within the melanophores of an isolated fish scale

The noradrenaline-induced pigment aggregation within the melanophores of the cuckoo wrasse ( Labrus ossifagus L.) has been shown to be mediated by postsynaptic alpha₂-adrenoceptors which in turn act via an inhibitory control of adenylate cyclase. In a previous paper it was shown that pertussis toxin (PT) caused a blockade of noradrenaline-induced pigment aggregation. Based on these findings, an assay for PT has been developed. The method involves toxin titration directly on isolated fish scales from L. ossifagus , takes about 2 h to perform and needs no sophisticated equipment. The method allows detection of femtogram quantities of toxin per ml. The effect of purified PT was neutralized by antiserum. Some preliminary results indicate that it is possible to detect PT-like activity in sputum samples from patients with suspected whooping cough, whereas no PT-like activity could be detected in samples from healthy volunteers.