A GENE AMPLIFICATION-HYBRIDIZATION SENSOR BASED METHODOLOGY TO RAPIDLY SCREEN AEROSOL SAMPLES FOR SPECIFIC BACTERIAL GENE SEQUENCES

摘要

The rapid detection of pathogenic microbial species in aerosols is of paramount importance considering its public health and animal husbandry implications. Oligonucleotide primers targeted against the DNA binding protein genes (hns) under appropriate amplification conditions is capable of specifically detecting Salmonela spp. and Clostridium spp. To demonstrate a methodology for rapidly detecting specific bacterial gene sequences, aerosol samples were collected from an outdoor biosolid application site and from indoor poultry houses for use in the protocol. Aliquots of these aerosol samples were used as templates in a gene amplification-hybridization sensor protocol to rapidly detect the DNA binding protein (hns) gene sequences. Using the combination of gene amplification and the hybridization sensor, the presence of the hns gene sequences was confirmed in the aerosol samples. The primary advantage of employing the hybridization sensor is that it confirms the sequence specificity of the amplified product within about 45 minutes, even if the product is present in extremely small concentrations. As little as 640 pg of amplified material could be rapidly confirmed using this hybridization sensor. The gene amplification and the confirmation of the amplification product's sequence can be completed in approximately 5 hours from the time the sample is collected. This study demonstrates that it is possible to rapidly detect and confirm the presence of specific bacterial genera in aerosols by combining gene amplification reactions with appropriate primers and post amplification dete