Application of 5'-nuclease PCR for Quantitative Detection of Listeria Monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 2000 Sep 30

PMID 11010869

Citations 46

Authors

H K Nogva

K Rudi

K Naterstad

A Holck

D Lillehaug

Affiliations

Soon will be listed here.

Abstract

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5'-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5'-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5'-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

Citing Articles

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopy Observation.

Brauge T, Bellay M, Midelet G, Soumet C Methods Mol Biol. 2024; 2852:33-46.

PMID: 39235735 DOI: 10.1007/978-1-0716-4100-2_3.

Real-time Fluorescent PCR Techniques to Study Microbial-Host Interactions.

Mackay I, Arden K, Nitsche A Methods Microbiol. 2024; 34:255-330.

PMID: 38620210 PMC: 7148886. DOI: 10.1016/S0580-9517(04)34010-9.

The Detection of Foodborne Pathogenic Bacteria in Seafood Using a Multiplex Polymerase Chain Reaction System.

Li P, Feng X, Chen B, Wang X, Liang Z, Wang L Foods. 2022; 11(23).

PMID: 36496717 PMC: 9736724. DOI: 10.3390/foods11233909.

Distribution and Characterization of Antimicrobial Resistant Pathogens in a Pig Farm, Slaughterhouse, Meat Processing Plant, and in Retail Stores.

Bae D, Macoy D, Ahmad W, Peseth S, Kim B, Chon J Microorganisms. 2022; 10(11).

PMID: 36422322 PMC: 9693440. DOI: 10.3390/microorganisms10112252.

Real-Time PCR Method Combined with a Matrix Lysis Procedure for the Quantification of in Meat Products.

Labrador M, Gimenez-Rota C, Rota C Foods. 2021; 10(4).

PMID: 33808357 PMC: 8066123. DOI: 10.3390/foods10040735.

References

Heid C, Stevens J, Livak K, Williams P . Real time quantitative PCR. Genome Res. 1996; 6(10):986-94. DOI: 10.1101/gr.6.10.986. View

McKillip J, Jaykus L, Drake M . Nucleic acid persistence in heat-killed Escherichia coli O157:H7 from contaminated skim milk. J Food Prot. 1999; 62(8):839-44. DOI: 10.4315/0362-028x-62.8.839. View

Holland P, Abramson R, Watson R, Gelfand D . Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991; 88(16):7276-80. PMC: 52277. DOI: 10.1073/pnas.88.16.7276. View

Raeymaekers L . Quantitative PCR: theoretical considerations with practical implications. Anal Biochem. 1993; 214(2):582-5. DOI: 10.1006/abio.1993.1542. View

Thompson J, Higgins D, Gibson T . CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22(22):4673-80. PMC: 308517. DOI: 10.1093/nar/22.22.4673. View

Mengaud J, Vicente M, Chenevert J, Pereira J, Geoffroy C, Baquero F . Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes. Infect Immun. 1988; 56(4):766-72. PMC: 259368. DOI: 10.1128/iai.56.4.766-772.1988. View

Pearson W, Lipman D . Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988; 85(8):2444-8. PMC: 280013. DOI: 10.1073/pnas.85.8.2444. View

Bassler H, Flood S, Livak K, Marmaro J, Knorr R, Batt C . Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl Environ Microbiol. 1995; 61(10):3724-8. PMC: 167671. DOI: 10.1128/aem.61.10.3724-3728.1995. View

Olsen J, Aabo S, Hill W, Notermans S, Wernars K, Granum P . Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. Int J Food Microbiol. 1995; 28(1):1-78. DOI: 10.1016/0168-1605(94)00159-4. View

10.

Gibson U, Heid C, Williams P . A novel method for real time quantitative RT-PCR. Genome Res. 1996; 6(10):995-1001. DOI: 10.1101/gr.6.10.995. View

11.

Rudi K, Kroken M, Dahlberg O, Deggerdal A, Jakobsen K, Larsen F . Rapid, universal method to isolate PCR-ready DNA using magnetic beads. Biotechniques. 1997; 22(3):506-11. DOI: 10.2144/97223rr01. View

12.

Chen S, Yee A, Griffiths M, Larkin C, Yamashiro C, Behari R . The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int J Food Microbiol. 1997; 35(3):239-50. DOI: 10.1016/s0168-1605(97)01241-5. View

13.

Kalinina O, Lebedeva I, Brown J, Silver J . Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res. 1997; 25(10):1999-2004. PMC: 146692. DOI: 10.1093/nar/25.10.1999. View

14.

Rudi K, Larsen F, Jakobsen K . Detection of toxin-producing cyanobacteria by use of paramagnetic beads for cell concentration and DNA purification. Appl Environ Microbiol. 1998; 64(1):34-7. PMC: 124668. DOI: 10.1128/AEM.64.1.34-37.1998. View

15.

Waters L, Jacobson S, Kroutchinina N, Khandurina J, Foote R, Ramsey J . Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal Chem. 1998; 70(1):158-62. DOI: 10.1021/ac970642d. View

16.

Sheridan G, Masters C, Shallcross J, Mackey B . Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells. Appl Environ Microbiol. 1998; 64(4):1313-8. PMC: 106147. DOI: 10.1128/AEM.64.4.1313-1318.1998. View

17.

Ibrahim M, Lofts R, Jahrling P, Henchal E, Weedn V, NORTHRUP M . Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal Chem. 1998; 70(9):2013-7. DOI: 10.1021/ac971091u. View

18.

Cheng J, Sheldon E, Wu L, Uribe A, Gerrue L, Carrino J . Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips. Nat Biotechnol. 1998; 16(6):541-6. DOI: 10.1038/nbt0698-541. View

19.

Norton D, Batt C . Detection of viable Listeria monocytogenes with a 5' nuclease PCR assay. Appl Environ Microbiol. 1999; 65(5):2122-7. PMC: 91307. DOI: 10.1128/AEM.65.5.2122-2127.1999. View

20.

Deneer H, Boychuk I . Species-specific detection of Listeria monocytogenes by DNA amplification. Appl Environ Microbiol. 1991; 57(2):606-9. PMC: 182759. DOI: 10.1128/aem.57.2.606-609.1991. View