Identification and enumeration of Listeria monocytogenes by nonradioactive DNA probe colony hybridization.

A plasmid containing the cloned listeriolysin gene of Listeria monocytogenes was used as a probe to identify Listeria strains by DNA colony hybridization. The probe DNA was labeled with horseradish peroxidase in the presence of glutaraldehyde. After the hybridization and wash procedures, the hybrid molecules were detected by luminescence, which resulted from the oxidation of luminol by a horseradish peroxidase-hydrogen peroxide-coupled reaction. Of the 150 Listeria strains and 16 non-Listeria strains examined, the probe hybridized only with L. monocytogenes. The technique was also used to enumerate L. monocytogenes in artificially contaminated foods.     

Listeria monocytogenes as a probe of immune function.

For almost half a century, the mouse model of Listeria monocytogenes infection has been used to analyse both innate and adaptive components of immunity and to discover key immune genes. Vast accumulated knowledge about the disease in mice provides a unique framework for identifying and characterising immune molecules using a variety of experimental approaches. To illustrate the range of questions that can be addressed using modern genetics and genomics tools, the authors provide an overview of the analysis of components of immune signalling networks using the mouse model of L. monocytogenes infection.     

Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization

A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe.

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.     

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.     

A cloned 23S rRNA gene fragment of Bacillus subtilis and its use as a hybridization probe of conserved character

Abstract A subclone of plasmid p14B8 containing the major part of a 23S rRNA gene of Bacillus subtilis was constructed and designated pJK1. Labeled plasmid pJK1 could be used as a DNA probe with conserved gene sequences. DNA-DNA hybridization experiments between filter-bound DNA from various bacteria and labeled pJK1 showed a good correlation between oligonucleotide sequence analysis of 16S rRNA and DNA homology values. Application of suboptimal or stringent hybridization conditions and an additional short incubation under the same conditions following hybridization yielded the best data for differentiating organisms related to B. subtilis from less or non-related bacteria.     

Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization.

A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.     

Genomic fingerprinting of microorganisms: its use as a hybridization probe of phage M13 DNA

Hypervariable nucleotide sequences detected by hybridization with the phage M13 DNA probe were found in the chromosomal DNAs of certain pathogenic microbial species. DNA fingerprinting, based on hybridization of M13-probe with hypervariable chromosomal DNA sequences, opens new approaches to epidemiological analysis, epidemiological prognosis, taxonomy, and other theoretical and applied fields of bacteriology.     

DNA carrying aliphatic amino groups and the use of its fluorescent derivative as a probe in molecular hybridization

A two-step method of labelling DNA through aliphatic amino groups with various reporter molecules has been developed. The method is based on reaction of cytosine-residues of polynucleotide chain with 4-aminooxybutylamine; the latter's synthesis is described. DNA modified on this way with fluorescein isothiocyanate functions a probe in molecular hybridisation. The sensitivity of this method for fluorochrome-labelled DNA probes in the dot hybridisation procedure is about 200 pg per mm2.     

The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy

Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.     

Dried gel hybridization in place of Southern hybridization for detection of Listeria monocytogenes DNA fragments

A simple, sensitive, dried gel DNA hybridization method for detection of Listeria monocytogenes DNA fragments is described. DNA samples were fractionated on an agarose gel. The gel was then denatured in NaOH-NaCl and neutralized in Tris-NaCl. The resulting agarose gel was dried and hybridized with 32P-labelled DNA probe. No transfer to nitrocellulose membranes was used.     

Rapid confirmation of Listeria monocytogenes isolated from foods by a colony blot assay using a digoxigenin-labeled synthetic oligonucleotide probe.

An oligodeoxyribonucleotide probe based on the sequence of a 321-bp internal fragment of the msp gene encoding a major secreted polypeptide of Listeria monocytogenes was labeled with digoxigenin by using terminal deoxynucleotidyl transferase. The specificity of the digoxigenin-labeled probe was determined by dot blot assays. The probe reacted with all strains of L. monocytogenes tested (12 of 12 strains representing five serotypes). The probe did not react with any other Listeria species or with other gram-positive bacteria (Brochothrix, Erysipelothrix, Corynebacterium, Rhodococcus, Lactobacillus, Leuconostoc, Bacillus, Staphylococcus, and Streptococcus). The probe was used to develop a colony blot assay for the rapid confirmation of L. monocytogenes on Listeria-selective agars which had been streaked with food enrichment cultures. Forty-eight food samples were tested by conventional culture and DNA colony blot assay. The sensitivity and specificity of the DNA colony blot were 100 and 97%, respectively.     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

A complex of single-strand binding protein and M13 DNA as hybridization probe.

Single-stranded DNA was complexed to the single-strand binding protein (SSB) of Escherichia coli in a mass ratio of 30:1. The protein moiety of this complex can be labelled by a number of methods of which we have chosen radio-iodination and biotinylation as examples. The SSB-M13 DNA complexes, labelled to high specific activities, were used as probes in hybridization experiments in which 1.6 X 10(-18) moles of immobilized target DNA were detected. The stability of the hybrids was not severely decreased by the binding of SSB. Analysis of hybrids by electron microscopy showed that complexing of DNA with SSB could be used to allow its subsequent identification in the hybrids.