Performance Assessment of the GENE-TRAK® Colorimetric Probe Assay for the Detection of Foodborne Salmonella spp.

A performance assessment of the GENE-TRAK® colorimetric probe assay using pure cultures and naturally contaminated foods and animal feeds underlined the high sensitivity and specificity of this DNA-rRNA diagnostic system. The probe effectively identified 110 (100%) strains of Salmonella spp. and yielded no false-positive reactions in the examination of 61 pure cultures of nonsalmonellae. Of the 53 contaminated raw meats and other high-moisture samples examined in this study, 47 (88.7%) were detected using the GENE-TRAK®analytical protocol. The system also identified 21 (95.5%) of 22 contaminated low-moisture foods and animal feeds. The overall sensitivity and specificity of the GENE-TRAK assay procedure with the naturally contaminated foods examined in this study was 90.7 and 100%, respectively. Ancillary work showed that the choice of selective enrichment conditions played a determinant role in the performance of the probe system. Although the GENE-TRAK protocol relies on varying temporal regimens of selective enrichment in tetrathionate brillant green (TBG35) and selenite cystine (SC35), and postenrichment in gram-negative (GN) broth for high and low-moisture food products, our results suggest that, irrespective of product type, probing of GN (6-h) postenrichment cultures inoculated from homologous TBG43 and SC35 (24-h) cultures and subsequent combination of these postenrichment cultures into a single probe assay would enhance the performance of the GENE-TRAK system to a level of unfailing sensitivity and specificity. Attempts at method brevity through direct probing of TBG43, TBG35, SC35, and Rappaport-Vassiliadis (RV43) enrichment cultures proved unsuccessful where probing of short (6-h) enrichment cultures identified ≤15.1 % and ≤72.7% of contaminated high- and low-moisture foods, respectively. Direct probing of 24 h enrichment cultures yielded homologous values of ≤17.0% and ≤77.3%. These findings suggest that components in the selective enrichment broths inhibit the probe assay. This negative effect was most prominent with RV43 enrichment cultures where most samples known to contain Salmonella spp. yielded false-negative reactions. Treatment of RV43 cultures, with 0.1 M sodium citrate prior to probe assay was partially effective in neutralizing the inhibitory agent(s).

Comparative study of colorimetric DNA hybridization method and conventional culture procedure for detection of Salmonella in foods.

A second generation nucleic acid hybridization assay has been developed and evaluated against the conventional culture method for detection of salmonellae in foods. The assay involves a liquid hybridization with Salmonella-specific oligonucleotide probes, capture of probe:target hybrids onto a solid support (plastic dipstick), and a colorimetric end point detection. The assay can be completed in 2.5 h, following approximately 44 h of culture enrichment. One thousand samples representing 20 food types were analyzed in parallel by both methods. Samples included uninoculated test product, and product inoculated with Salmonella at 2 levels. Eighteen Salmonella serotypes were used as inocula. The data demonstrate that the colorimetric hybridization method and the conventional culture method are equivalent in their ability to detect Salmonella contamination of foods.

Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study.

A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.

DNA hybridization assay for detection of Salmonella in foods: collaborative study.

A collaborative study was performed in 11 laboratories to validate a DNA hybridization (DNAH) procedure for detection of Salmonella in foods. The DNAH procedure was compared to the standard culture method for detection of Salmonella in 6 foods: ground pepper, soy flour, dry whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. With the exception of turkey which was naturally contaminated, uninoculated and inoculated samples of each food group were analyzed. Results for the DNAH method were significantly better than for the standard culture method at the 5% probability level for the detection of Salmonella in turkey. There was no significant difference between the methods for the other 5 foods. The method has been adopted official first action.

A phi 80 function inhibitory for growth of lambdoid phage in him mutants of Escherichia coli deficient in integration host factor. I. Genetic analysis of the Rha phenotype.

Bacteriophage phi 80 and lambda-phi 80 hybrid phage of the type lambda (QSR)80, in which the rightmost 10% of the lambda genome is replaced by corresponding phi 80 material, are unable to grow lytically in himA and hip/himD mutants of Escherichia coli K12 at 32 degrees. The genetic element responsible for the growth defect, rha, has been mapped to the (QSR)80 region and was located more precisely by restriction enzyme and DNA heteroduplex analysis of mutations that result in loss of the Rha phenotype. Such an Rha mutant carrying a 1.5-kb deletion beginning 0.58 kb from the right end of the chromosome and extending leftward locates the rha locus at least in part within this region of (QSR)80. In addition, a substitution derivative of lambda (QSR)80 was isolated which does not exhibit the Rha phenotype. In this phage, lambda-80hy95, the right half of the (QSR)80 region is replaced by DNA homologous to the 95-100% segment of lambda. In mixed infections in the himA42 host at 32 degrees, lambda + does not complement lambda (QSR)80 for growth and the burst size of the coinfecting lambda + is reduced in comparison to that in a single infection. Deletion mutants of lambda (QSR)80 that grow normally in himA42 at 32 degrees in single infections are inhibited for growth in mixed infections with lambda (QSR)80. These results suggest the existence of a trans-acting function which inhibits phage growth in the absence of HimA or Hip/HimD function. It is likely that the rha gene either encodes that function or indirectly controls its action.

A phi 80 function inhibitory for growth of lambdoid phage in him mutants of Escherichia coli deficient in integration host factor. II. Physiological analysis of the abortive infection.

Derivatives of phage lambda with the rightmost 3% of the genome (the QSR region) from the related phage phi 80 fail to grow at low temperatures (e.g., 32 degrees) in Escherichia coli hosts deficient in either protein component of IHF (integration host factor), the products of the himA and hip/himD genes. The abortive infection of lambda (QSR)80 in mutants defective for IHF was studied in detail. This infection is characterized by a lack of cell lysis and an inhibition of phage DNA replication after an initial period of normal synthesis. An inhibition of host DNA replication also occurs after a similar period of apparently normal synthesis, and the abortive lambda (QSR)80 infection is lethal to the host. An assay of beta-galactosidase activity in lambda (QSR)80-infected cells provided indirect evidence that RNA and protein synthesis continue late into the abortive infection. The defective growth is imposed by the product of the rha gene located in the (QSR)80 genetic material. Two-dimensional electrophoretic analysis of phage proteins produced in ultraviolet (uv)-irradiated phage-infected host cells has demonstrated the existence of a protein that is encoded or whose synthesis is regulated by the rha locus. Based on these findings, possible roles for a HimA-Hip/HimD-controlled rha product in a late stage of phi 80 development are discussed.

Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.

Mutations reducing the activity of c17, a promoter of phage lambda formed by a tandem duplication.

We report the isolation of four independently selected mutations (scs) in the c17 promoter of phage lambda that reduce or eliminate the promoter activity. The c17 promoter is not normally present in lambda, and has been shown to be generated by a tandem duplication which creates a "Pribnow Box," a heptamer sequence implicated in promoter activity. This sequence is located upstream from the site of transcription initiation and is present, with some variation, in all promoters whose sequences have been determined. Analysis of the c17 duplications carrying the scs mutations reveals that three of these mutants carry single base-pair changes in the most highly conserved base pairs of the Pribnow Box and that the other mutation is a reversion to the wild type sequence in this region (i.e., a loss of the duplicated base pairs).