Polymerase Chain Reaction (PCR) Random Amplified Polymorphic DNA (RAPD) primer design for the detection of Listeria monocytogenes

Date

4-2010

Degree

Bachelor of Science in Biology

College

College of Agriculture and Food Science (CAFS)

Adviser/Committee Chair

Joel C. Mendoza

Co-adviser

Francisco B. Elegado

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Abstract

The development of PCR-based detection kits for food-borne pathogens have been underway since their usefulness in detecting and preventing food contamination has been realized. One such pathogen is Listeria monocytogenes which causes listeriosis. L. monocytogenes reference strains (Lm1, Lm2, Lm3 and Lm4) and Enterococcus strains (027 and 064) isolated from different food samples were obtained from the Food and Feed Laboratory, BIOTECH, UPLB. DNA isolation was done though the CTAB method and the isolated DNA was subjected to amplification of iap and Lis3 genes, via polymerase chain reaction and the use of iap and Lis3 primers, for the detection of L. monocytogenes. Detection of the genes correlated with the cultural identification previously done for L.monocytogenes Lm1, Lm2, Lm3, and Lm4. Different RAPD primers were used to screen for a unique band, which can serve as a specific molecular marker for L.monocytogenes. Gene amplification using primer 066 resulted in a potential specific marker 1.2 kb in size. Molecular cloning of the isolated RAPD marker for L.monocytogenes was done to facilitate sequencing of the amplicon. Twenty-three white colonies representing presumptive positive clones were subjected to plasmid DNA extraction and restriction enzyme analysis to determine the presence of the target insert fragment. The two plasmid clones confirmed the presence of the target 1.2 kb insert fragment through hybridization. The plasmid clone (5a1) was sent for sequencing at Macrogen, Korea using the universal primers, M13. Multiple sequence alignment analysis of the 950-base marker sequence using BLAST-N revealed 99% to 100% homology with L. monocytogenes. Primer design of the molecular marker was done through Primer3 Plus primer design software. The primers designed had good melting temperatures and GC content. However, secondary structures such as hairpins and self- dimers were observed. Thus, verification of the successful primer design should be tested upon its actual run on PCR using L. monocytogenes as the template DNA. The most significant contribution of this study is the development of a PCR-based kit for detection and identification of L.monocytogenes in different food samples.

Language

English

Location

UPLB Main Library Special Collections Section

Document Type

Thesis

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