Design of Optimized PCR Method for Detection of SHV Type ESBL Genes

ESBL is an important factor in antibiotic resistance. Antibiotic resistance has been proposed as a global problem in recent decades and it causes many deaths all over the world; therefore, rapid and accurate diagnosis can be major step in solving this problem. This study aimed to identify SHV-type gene with PCR method.
Methods and Materials: SHV gene sequences were extracted and aligned from Genbank. Specific PCR primers were designed according to the consensus of the SHV genes. A plasmid containing positive control of the gene was created by using PCR and cloning methods. The assay specificity was evaluated using Brucella genomic DNA and a panel containing genomes of 10 gram-positive and gram-negative organisms. The PCR assay was highly specific and no amplification products were observed from the non-SHV organisms, an other test of the specifity was done by the use of CTX-M and TEM betalactamases.
A band with a the length of 199bp was indicated in the agarose gel electrophoresis of the PCR product in accordance with the desired sequence. Showing the same fragment in PCR confirmed the recombinant plasmid of coloning product containing the SHV gene. When the plasmid was subjected to the SHV-PCR, a ladder-like pattern was visualized on the agarose gel. The pattern has never observed in the case of negative controls.The results of specify assay conveyed that primers were separately for SHV gene.
Discussion and The results of this study indicated that PCR assay is a simple, rapid, sensitive and specific technique for detection of SHV gene that may improve diagnostic potential in clinical laboratories.