Abstract
Molecular methodologies are fast becoming preferred detection methods for pathogen analysis. Techniques such as polymerase chain reaction (PCR), microarrays, nucleic acid sequence based amplification (NASBA) have taken the lead over microbiological detection methods. Factors that have given molecular techniques the added advantage are the reduced detection time, ease of performance, increased sensitivity of assays and easy interpretation of results. Currently a variety of PCR assays exist for the successful amplification of various bacterial, viral and protozoan nucleic materials. Some assays are commercially available for use in routine testing of pathogens from water, faecal and food sources. The nucleic material of the pathogen is extracted from the source and amplified using a specific PCR protocol. The amplified PCR products are confirmed/ visualized by gel electrophoresis. Gel electrophoresis enables end-point detection of PCR products; however, it entails restrictions such as the time taken for the preparation of agarose gels by trained personnel, use of ethidium bromide (carcinogenic) for visualizing the PCR products and its immobility for analysis in point of care use. Hence with the focus on rapid confirmation of PCR products and point of care testing the possible use of LFIA for confirmation was investigated.
A sandwich format LFIA was developed to enable detection of the amplified PCR products (Horng et. al., 2006). The test strip comprised of a nitrocellulose membrane with the immobilized tag specific antibodies, a sample pad for the application of the amplified PCR products mixed with running buffer, a conjugate pad soaked with gold nanoparticles for visualization and an absorbent pad to remove the excess sample from the test strip .The test strip detects the presence of amplified PCR products using primers labelled with two different tags. The test strip is labelled with the tag specific antibody and recognition occurs upon conjugation of the tag and tag specific antibody. The complex is visualized when it conjugates to coloured particles such as gold nanoparticles which produce a red line on the test strip.
LFIA test strips can successfully detect Vibrio, Salmonella, entero invasive E. coli (EIEC) and Shigella species from various samples. The assay had also been validated in terms of its specificity, sensitivity, repeatability and reproducibility and produced satisfactory results for each of the assay validation components. This detection/confirmation method could assist greatly in the rapid detection of pathogens...
D.Tech. (Biomedical Technology)