A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences

Blais, Burton W.; Turner, G. S.; Sooknanan, Roy; Malek, Lawrence T.

doi:10.1128/aem.63.1.310-313.1997

Cited by 59 publications

(27 citation statements)

References 12 publications

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“…While widely used in the clinical realm, particularly for diagnosis of viral infection and determination of viral load in clinical specimens, the application of NASBA to the detection of food-borne bacterial pathogens is relatively new. NASBA has been reported for the detection of the Norwalk-like viruses (Greene et al 2003), hepatitis A virus (Jean et al 2002), S. enterica (Simpkins et al 2000), L. monocytogenes (Blais et al 1997) and C. jejuni (Uyttendaele et al 1997). However, only two of these studies evaluated the method in a food matrix.…”

Section: Discussionmentioning

confidence: 99%

“…The NucliSens Ò Basic Kit provides reagents and protocols for semiautomated RNA extraction, NASBA amplification and ECL detection of amplicons, all of which can be completed within 8 h. Similar methods are in widespread use in clinical laboratories for the detection of HIV (Kievits et al 1991) and cytomegalovirus (Greijer et al 2002). Although NASBA applications are in use for the detection of food-borne pathogens including the Norwalklike viruses (Greene et al 2003), hepatitis A virus (Jean et al 2002), Salmonella enterica (Simpkins et al 2000), Listeria monocytogenes (Blais et al 1997) and Campylobacter jejuni (Uyttendaele et al 1997), only a few have applied the method to specific food matrices (Blais et al 1997;Cook et al 2002). Using a previously reported NASBA-ECL protocol designed to detect mRNA transcripts from the bacterial Hsp70 homologue dnaK gene of S. enterica (Simpkins et al 2000), the purpose of our study was to determine the feasibility of using this NucliSens Ò Basic kit NASBA-ECL protocol for the rapid and sensitive detection of S. enterica from a variety of representative food commodities.…”

Section: Introductionmentioning

confidence: 99%

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Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

D’Souza

Jaykus

2003

J Appl Microbiol

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Aims: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. Methods and Results: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens Ò Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 10 1 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. Conclusions: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 10 2 and 10 1 CFU per 25 g food sample.Significance and Impact of the Study: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

D’Souza

Jaykus

2003

J Appl Microbiol

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“…Studies where the hlyA transcript has been used as marker for viable or dead bacteria (Klein and Juneja 1997;Norton and Batt 1999), or as a mean for the detection of L. monocytogenes (Blais et al 1997) have also been carried out. From the knowledge gained in this work, such applications could be difficult as the absence of gene expression under the conditions tested could be misinterpreted as the absence of L. monocytogenes, or the presence of dead bacteria.…”

Section: Diverse Gene Expression Patternsmentioning

confidence: 99%

Subtyping Listeria monocytogenes through the combined analyses of genotype and expression of the hlyA virulence determinant

Rudi¹,

Nogva²,

Naterstad³

et al. 2003

J Appl Microbiol

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Aims: A major challenge for Listeria monocytogenes diagnostics is that this bacterium is ubiquitous in the environment, and that only a small fraction of the lineages are potential human pathogens. The aim of this work was to obtain a better subtyping of L. monocytogenes through utilization of combined analyses of genotype and the expression of the virulence determinant hlyA. Methods and Results: We investigated the effect of growth temperature and medium on the hlyA expression. The gene expression levels were determined by real-time quantitative reverse transcription PCR. The expression pattern of hlyA was highly diverse among the different strains tested. The expression ranged from repression to a 1000-fold induction for growth at 42°C, as compared with 0°C. The expression patterns were compared with the corresponding genotypes. There were surprisingly low correlations between the expression patterns and the genotype clusterings.This is exemplified for the virulent type strain NTNC 7973 and non-virulent type strain DSMZ 20600. These strains are genetically nearly identical, while the hlyA gene expression patterns are very different. Conclusions: The hlyA gene expression was highly diverse even within genetically clustered subgroups of L. monocytogenes. Consequently, the gene expression patterns can be used to further differentiate the strains within these genetic subgroups. Significance and Impact of the Study: A major limitation in the control of L. monocytogenes is that the current tools for subtyping are not accurate enough in determining the potential virulent strains. The impact of this study is that we have developed a subtyping approach that actually targets a virulence property.

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“…Its advantage over the traditional RT-PCR technique is its suitability for the direct ampli¢cation of RNA targets, obviating the need for thermal cycling equipment. NASBA was successfully used for the detection of Listeria monocytogenes [6], Campylobacter [7] and human immunode¢ciency virus [8]. In recent work, we have demonstrated the e¡ectiveness of NASBA for the detection of hepatitis A virus in wastewater, fruits and vegetables [9].…”

Section: Introductionmentioning

confidence: 97%

Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)–enzyme-linked immunosorbent assay in sewage treatment effluent

Jean¹

2002

FEMS Microbiology Letters

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A colorimetric nucleic acid sequence-based amplification^enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for rapid detection and identification of human rotavirus. Oligonucleotide primers targeting gene 9 encoding a serotype-specific antigen VP7 were selected and used for the amplification of viral RNA by the isothermal NASBA process, resulting in the accumulation of biotinylated RNA amplicons. Amplicons were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA^RNA hybrids were colorimetrically detected by the addition of streptavidin^peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4U10 1 PFU ml 31 ) and 15 PFU (3U10 3 PFU ml 31 ) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. No interference was encountered in the amplification and detection of rotavirus in the presence of non-target RNA or DNA. Moreover, the presence of nontarget bacteria and virus does not generate any non-specific signal, confirming the specificity of the developed NASBA-ELISA system and its effectiveness in specifically detecting rotavirus. The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples. ß

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A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences

Cited by 59 publications

References 12 publications

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

Subtyping Listeria monocytogenes through the combined analyses of genotype and expression of the hlyA virulence determinant

Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)–enzyme-linked immunosorbent assay in sewage treatment effluent

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