Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10⁴ cfu/ml (phenyl phosphate system) or 10⁵ cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1–5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).     

Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes.

Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3' region of L. monocytogenes hlyA gene spanning a conserved HindIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10(4) cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10-100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days.     

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3'region of L. monocytogenes hly A gene spanning a conserved Hin dIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10⁴ cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10–100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred.     

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred. and accepted 22 June 1989     

A semi-homogeneous amperometric immunosensor for protein A-bearing Staphylococcus aureus in foods

Summary A semi-homogeneous amperometric immunosensor specific to the protein A of Staphylococcus aureus was developed using direct electrochemical detection of phenol produced by alkaline phosphatase from phenyl phosphate. The immunosensor could reliably detect strains of protein A-bearing S. aureus in pure cultures at ca. 30⁴ cfu/ml, and at ca. 10⁵ cfu/g or ml in various food samples. Due to its semi-homogeneous nature, the system was very simple, easy to operate, and labour-saving. The good correlation between the amperomatric current generated by the immunosensor and plate counts illustrated the potential usefulness of this simple system. It proved to be a reliable 24-h detection method for food samples containing very low numbers of protein A-bearing S. aureus after pre-enrichment, as it was able to detect cells that could not directly be enumerated by plate counts.Offprint requests to: R. G. Kroll     

Efficiency of Selenite Cystine and TT Enrichment Broths for the Detection of Salmonella

Comparisons of selenite cystine (SC) and TT enrichment broths for detecting salmonellas were made with pure culture suspensions, with samples of naturally or artificially contaminated foods and with poultry feed. Selenite cystine recovered higher numbers of salmonellas from pure cultures and ground beef, while TT broth recovered higher numbers from pork sausage and poultry feed. Differences in the recovery of salmonellas from other food products appeared to be insignificant. The use of both SC and TT is thus recommended for maximal recovery of these organisms.     

鼠伤寒沙门氏菌多重PCR检测方法的研究

分别根据沙门氏菌16S rRNA、质粒毒力基因spvC、致病基因invB、fimA序列设计4对引物,对沙门氏菌株及非沙门氏株菌基因组DNA进行多重PCR检测。结果该方法能检测出6.3×10² 个cfu/ml纯培养的沙门氏菌,人工染菌食品模拟检测结果显示,熟鸡肉初始含菌量为17cfu/g、全脂奶粉为11cfu/g、生牛肉为13.6cfu/g,经过8h增菌,PCR检测为阳性。该体系能鉴定产生多种毒力因子的沙门氏菌,特异性强、敏感性高,为检测和鉴定沙门氏菌株提供了一个新方法。     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas

D.CHOI, R.S.W. TSANG AND M.H. NG. 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10⁶/ml of a smooth wild strain of Salm. typhimurium , and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.     

Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10⁸/ml or more. During incubation in TBB at 43C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10³–10⁷/ml in BPw and after transfer to TBB slowly reached 10⁴/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5×10⁵ cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10⁶ cfu/ml.     

RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 10³ 10⁵ cfu/mL, with R²= 0.99. The detection limit of this method was 1.09 × 10³ cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.