Optimization of Multiplex PCR Assays to Identify Genes Involved in Tetracycline Resistance
Abstract
Antibiotics play a vital role in modern life including animal husbandry and healthcare, but the chronic use of antibiotics has lead to an increase in antibiotic resistance among bacteria. During treatment with antibiotics, bacteria without resistance subside, leaving only those with antibiotic resistant genes. The presence of antibiotic resistant genes in these organisms can be profiled, but this requires multiple steps, including preparing the master mix, performing PCR (polymerase chain reaction), and running gel electrophoresis to determine the products. Multiplex PCR (Ng; Fan) simplifies the process by combining primer sets for multiple genes within the same reaction, allowing 4-5 genes to be tested per reaction versus a single gene at a time. This project will attempt to simplify the PCR process by combining primer sets for 4-6 genes within a single reaction, instead of amplifying each gene individually. This will increase the number of bacterial samples that can be analyzed and further reduce extraneous variables such as improper technique. Tetracycline resistant bacteria will be grown from local soil samples, and the bacterial colonies will be analyzed for nineteen specific tetracycline resistant genes. To determine the effectiveness of this technique, PCR and gel electrophoresis will be completed on multiple combined samples and compared to single gene amplification. We should be able to identify individual DNA bands for each gene in the 4-5 gene reactions.
Optimization of Multiplex PCR Assays to Identify Genes Involved in Tetracycline Resistance
Conference Room C
Antibiotics play a vital role in modern life including animal husbandry and healthcare, but the chronic use of antibiotics has lead to an increase in antibiotic resistance among bacteria. During treatment with antibiotics, bacteria without resistance subside, leaving only those with antibiotic resistant genes. The presence of antibiotic resistant genes in these organisms can be profiled, but this requires multiple steps, including preparing the master mix, performing PCR (polymerase chain reaction), and running gel electrophoresis to determine the products. Multiplex PCR (Ng; Fan) simplifies the process by combining primer sets for multiple genes within the same reaction, allowing 4-5 genes to be tested per reaction versus a single gene at a time. This project will attempt to simplify the PCR process by combining primer sets for 4-6 genes within a single reaction, instead of amplifying each gene individually. This will increase the number of bacterial samples that can be analyzed and further reduce extraneous variables such as improper technique. Tetracycline resistant bacteria will be grown from local soil samples, and the bacterial colonies will be analyzed for nineteen specific tetracycline resistant genes. To determine the effectiveness of this technique, PCR and gel electrophoresis will be completed on multiple combined samples and compared to single gene amplification. We should be able to identify individual DNA bands for each gene in the 4-5 gene reactions.