Objective: To design the primers used for the detection of beta-lactam antibiotic resistance genes SHV, CTX-M, TEM a specific and the lowest cost. Subject and method: The sequences of the genes TEM, SHV, CTX-M were loaded from bank antibiotic resistance genes (http://www.lahey.org/studies/temtable.htm). Result and conclusion: In this study, the authors demonstrated a concept that deoxyinsosine using oligonucleotide sequences not found in nature, multi-segment captured in the conservation area of their low degeneration gene, can be used as primers to amplify all members with similar sequences of their genes and thus only once multiplex PCR assay can identify beta-lactam resistant caused by genes SHV, TEM, CTX-M. The primers were optimized for gene family SHV, CTX-M, TEM was found and PCR primers corresponding majority were built to allow detection of beta-lactam antibiotic resistance due to their genotypes under 3 genes above in a single PCR reaction tube.