DNA-DNA hybridization assay for detection of Salmonella spp. in foods

We have developed a DNA-DNA hybridization test for the presence of Salmonella spp. in foods. This test requires an initial pre-enrichment of food samples in nutrient broth but does not require selective enrichment. Samples of food cultures are collected on membrane filters and assayed by molecular hybridization to labeled probes. The probes consist of DNA sequences which are unique to the genus Salmonella and are widely distributed in the genus. A diverse panel of foods was assayed successfully by this methodology.     

Automated detection of Salmonella spp. in foods.

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.     

食品中沙门氏菌分子检测靶点的筛选与评价

[目的]发掘新的沙门氏菌分子检测靶点,筛选检测性能优秀的引物.[方法]利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性,发掘出100多个检测沙门氏菌属的特异性片段,并从中随机挑选出15个片段作为候选靶点,一共设计了27对引物(FS1～FS27),对它们的特异性、灵敏度加以评价,从中筛选检测性能最好的引物.[结果]在27对引物中,检测性能最优的引物为FS23,采用该引物对供试菌株的相应检测靶点进行PCR扩增,44株沙门氏菌都能扩增到一条492 bp特异性片段,而22株非沙门氏菌则不能扩增出这一特异性片段.以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA的灵敏度为11.9 fg/μL,细菌纯培养物灵敏度为4.9×102cfu/mL;用猪霍乱沙门氏菌人工污染牛奶样品,如果接种起始菌量为100 cfu/25 mL时,只需要增菌5 h,采用上述方法即能检测出沙门氏菌.[结论]引物FS23对应的基因序列是一个性能优良的新分子检测靶点,具备很高的特异性和灵敏性,能够广泛应用于食品中沙门氏菌的快速检测.     

A rapid monitoring assay for the detection of Salmonella spp. and Salmonella Senftenberg strain W775 in composts

AIMS: The composting process needs to be validated for its hygienic status in order to ensure that it is free of pathogens. Generally, this is evaluated through temperature monitoring, or additionally through active inoculation and monitoring of indicator organisms. We aimed to develop a monitoring method for the heat-resistant indicator organism Salmonella enterica ssp. enterica serovar Senftenberg strain W775 for detection in composting biowastes. METHODS AND RESULTS: The method development is comprised of: (i) optimization of molecular detection of bacteria belonging to the genus Salmonella; (ii) identification of a DNA marker for Salmonella strain W775; and (iii) development of a multiplex polymerase chain reaction (PCR)-based on both DNA markers. Subsequently, Salmonella strain W775 was inoculated and monitored during composting of biowastes in an industrial composting facility. CONCLUSIONS: A highly sensitive and specific detection of viable cells was obtained by enriching the compost sample prior to multiplex PCR analysis. Complete inactivation of Salmonella strain W775 was obtained within 4 days in an industrial composting facility at temperatures ranging between 41 and 57 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a monitoring method for the simultaneous detection of naturally occurring Salmonella strains and artificially introduced Salmonella strain W775 in composting biowastes that can be applied in routine analysis.     

Rapid radiometric method for detection of Salmonella in foods.

A radiometric method for the detection of Salmonella in foods has been developed which is based on Salmonella poly H agglutinating serum preventing Salmonella from producing 14CO2 from [14C]dulcitol. The method will detect the presence or absence of Salmonella in a product within 30 h compared to 4 to 5 days by routine culture methods. The method has been evaluated against a routine culture method using 58 samples of food. The overall agreement was 91%. Five samples negative for Salmonella by the routine method were positive by the radiometric method. These may have been false positives. However, the routine method may have failed to detect Salmonella due to the presence of large numbers of lactose-fermenting bacteria which hindered isolation of Salmonella colonies on the selective agar plates.     

Efficacy of a commercial polymerase chain reaction-based assay for detection of Salmonella spp. in animal feeds

Salmonellosis is a cyclic problem in the food industry, to which animal feed has been contributory. Current conventional methods of Salmonella spp. detection require 96 h for detection and confirmation. With modern and just-in-time production schedules, a 96-h hold represents a significant expense in storage and decontamination. The commercially available assay, 'BAX for Screening/Salmonella' (BAX), is based on the principle of the polymerase chain reaction and may represent a significant decrease in assay time. Seven fresh feed formulations, two fresh feed ingredients, seven stored feeds and two stored feed ingredients were artificially contaminated with a primary poultry isolate of Salmonella typhimurium and analysed by conventional and BAX methodology. The results of BAX agreed with conventional plating results for 16 of 18 samples spiked with 1200 cfu 10 g(-1) of feed and 13 of 18 samples spiked with 40 cfu 10 g(-1) of feed. Indigenous Salmonella spp. were detected in five of eight samples of poultry diets by conventional methods. With BAX, Salmonella spp. could not be detected in any of the samples after only 7 h of enrichment but could be detected in two dietary samples after 13 h of enrichment and four dietary samples after 24 h of enrichment. Specific sequences of salmonella DNA that were extracted from poultry diets could be detected with BAX.     

Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization

Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n?= 24) and (2) after storage at -20?°C for 6 months (n?= 24), (3) for 12 months (n?= 24), and (4) for 24 months (n?= 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p?< 0.001). No differences were found between groups 1 and 2 (p?> 0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.     

Twelve-hour PCR-based method for detection of Salmonella spp. in food

A PCR-based method for the detection of Salmonella spp. in food was developed. The method, set up on typical salami from the Italian region of Marche, is sensitive and specific and shows excellent correlation with the conventional method of reference when naturally contaminated foods are analyzed. Moreover, it can be easily performed within a maximum of 12 h from food sampling, thus allowing prompt detection of Salmonella spp. in the food stocks analyzed.     

Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

DNA-DNA hybridization in the systematics of Streptomyces.

The DNA relatedness among strains in several different phenotypically defined Streptomyces species clusters was evaluated. It was found that the data from DNA-relatedness studies do not necessarily agree with the clustering generated using numerical taxonomic techniques. A study of the morphologically heterogeneous 'S. cyaneus' cluster showed that morphological criteria traditionally used to classify and identify Streptomyces species still have value, since strains in DNA-relatedness cluster groups were also similar morphologically (i.e., they had similar spore color, surface properties, and sporophore morphology). An evaluation of DNA relatedness among strains in the S. violaceusniger and S. lavendulae clusters indicated that, if anything, the genus is underspeciated, based on the number of single-member clusters observed. A study of strains of the sweet potato pathogen, S. ipomoea, collected in various locations in the United States and Japan indicated, not surprisingly, that all of the strains belong to the same species.     

南方食品中沙门氏菌污染调查及分型

摘要:【目的】系统调查我国南方代表性地区食品中沙门氏菌污染情况,并对分离株进行血清分型和肠杆菌基因间重复共有序列聚合酶链式反应(Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction,ERIC-PCR)分型研究,为溯源和控制食品中沙门氏菌的污染提供数据。【方法】采用定性法和最大可能数(Most Probable Number,MPN)法对七大类食品(肉与肉制品、水产品、速冻食品、熟食、蔬菜、乳制品和食用菌)中的沙门氏菌进行检测。利用血清分型和分子分型确定南方沙门氏菌血清型的分布和优势血清型,研究分离株的遗传多样性。【结果】从400份食品样品共检出75 份沙门氏菌阳性样品,污染率为18.8%(75/400)。其中,97.3%(73/75)的阳性样品污染水平均低于10 MPN/g。对93株分离株进行血清分型,分属9个群、29种血清型。优势血清型为德尔卑沙门氏菌(Salmonella Derby)和鼠伤寒沙门氏菌(Salmonella Typhimurium)。利用ERIC-PCR对96株沙门氏菌(包括93株分离株和3株标准株)进行分型研究发现,在相似系数为0.75时可分为15个聚类簇,56种型别,分离株的基因型别呈现多样性。【结论】南 方食品中沙门氏菌的污染率较高,但污染水平低。S. Derby和S. Typhimurium为优势血清型。初步建立了沙门氏菌ERIC-PCR指纹图谱数据库,南方食品中沙门氏菌的血清型和基因型呈现多样性。     

A combined polymerase chain reaction-colour development hybridization assay in a microtitre format for the detection of Clostridium spp.

We have developed a rapid and sensitive assay for the detection of clostridial cells or spores in liquid food samples. The method recognizes Clostridium 16S (small ribosomal subunit RNA) rDNA (ribosomal DNA) sequences by a polymerase chain reaction-digoxigenin-labelling protocol, coupled to a capture oligonucleotide immobilized on a microtitre plate. The positive results are revealed by means of a colour reaction. In 6 h of non-intensive labour, we can detect as few as two to five clostridial cells or spores in experimentally contaminated soft drinks. Correspondence to: J. L. Ramírez     

Complementary DNA-DNA hybridization in Drosophila

Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.On leave from the Dipartimento di Biologia, II Università di Roma Tor Vergata, Rome, Italy     

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples.

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.     

Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods.

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.     

A modified conductance medium for the detection of Salmonella spp.

Selenite-Cystine/trimethylamine oxide/dulcitol medium has been used in conjunction with conductance instruments to detect the presence of Salmonella spp. in foods and faeces. However, a small but significant number of salmonella strains were missed by this method. The majority of these strains were detected when dulcitol was substituted by mannitol and tested on two separate Malthus conductance instruments. Some strains of Citrobacter freundii and Escherichia coli continued to give false positive results. Attempts are made to explain why the substitution of mannitol for dulcitol gives an improved medium.     

Evaluation of a fluorogenic assay for detection of Escherichia coli in foods.

A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods. Fluorescence is indicative of the presence of E. coli; extensive biochemical confirmation is unnecessary with this assay. The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E. coli on 270 samples of raw ingredients and powdered food products. Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives. We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive.     

Slow rehydration for detection of Salmonella spp. in feeds and feed ingredients.

Rehydration and equilibration (4 h) of feeds and feed ingredients at water/sample ratios (vol/wt) of 1.4 to 3.2 did not markedly increase recovery of Salmonella spp. in the slurry when analyzed by standard cultural and direct enrichment methods. Of 143 naturally contaminated samples examined, equilibration increased levels of detection from 106 to 109 positive samples by the standard cultural method and from 103 to 112 by direct enrichment. Results suggest that nonhomogeneous distribution of low incident numbers of salmonellae in test samples rather than an equilibration-dependent response provided for the observed heterogeneity in recovery patterns. The novel equilibration approach is of limited application and requires validation on an individual food basis.     

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10-100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.