Development of rapid detection methods for Listeria monocytogenes in foods and paraffinized human tissues using nonisotopic DNA-DNA hybridization techniques and polymerase chain reaction

Changmin Kim, Purdue University

Abstract

The hybridization characteristics of an internal fragment (pRF106 fragment) of the msp gene encoding a 60 kilodalton major secreted polypeptide were determined. An internal fragment designated pRF106 fragment hybridized only to a 13 kilobase-pair (kb) EcoRI fragment of L. monocytogenes and a 3 kb EcoRI fragment of 1 of 8 strains of L. seeligeri (F4882; cheese isolate) under stringent hybridization conditions (Tm-5$\sp\circ$C). The pRF106 fragment was used to develop a nonisotopic colony hybridization assay to detect L. monocytogenes on lithium chloride phenylethanol-moxalactam (LPM) plates. The nucleotide sequence of the pRF106 fragment was determined by the Sanger dideoxy sequencing method. Using the sequence information, 4 oligonucleotides were synthesized and their specificities were evaluated. One oligonucleotide (Msp110) was specific for L. monocytogenes in dot blot assays under hybridization conditions. Msp110 did not hybridize to DNA from L. seeligeri F4882 which was a problem when the pRF106 fragment was used as a probe. Msp110 was labeled with digoxigenin using terminal transferase and used in a colony hybridization assay to detect L. monocytogenes on LPM plates. This colony hybridization assay was evaluated against enrichment cultures of 66 naturally contaminated foods. Its sensitivity and specificity were 100% and 97% respectively. An internal fragment (734 bp fragment) of the gene coding for beta-hemolysin of L. monocytogenes serotype 1/2c (hlyA) was amplified by PCR and used as a probe in a chemiluminescent colony hybridization assay to detect L. monocytogenes on LPM plates streaked with naturally contaminated foods. The sensitivity and specificity of the method were 89% and 90%, respectively. The polymerase chain reaction (PCR) technique was applied to detect L. monocytogenes in Paraffinized human tissues by amplifying an internal fragment of the structural gene (hlyA) encoding the beta-hemolysin of L. monocytogenes. A nested PCR was used to decrease contamination problems and to amplify the diagnostic fragment (165 bp) which was visualized by agarose gel electrophoresis. The method was specific for L. monocytogenes and sensitive enough to detect 3 cells of L. monocytogenes. The method was applied to detect L. monocytogenes in placental and fetal tissues from suspect perinatal listeriosis cases.

Degree

Ph.D.

Advisors

Swaminathan, Purdue University.

Subject Area

Food science|Molecular biology

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