Abstract: To investigate a method which can rapidly and accurately detect the genotyping extended-spectrum beta-lactamases(ESBLs)-producing Gram-negative bacterium.  Methods　　Clinical isolated ESBLs-producing strains were detected by double-disk method. SHV gene fragments of ESBLs were amplified by polymerase chain reaction（PCR）. Pyrosequencing was used to study SHV genotyping of the 29 strains of ESBLs-producing Escherichia coli and Klebsiella pneumoniae isolated from hospitals in Chengdu. The gene polymorphism of amino acids site encoding 35 and 43 of SHV gene fragments were investigated. Meanwhile,antimicrobial susceptibility was determined by disk diffusion method.  Results　The results of pyrosequencing were that 21 of the 29 locally isolated ESBLs-producing strains had SHV gene fragments. Codon 43 amino acids had no polymorphism，while codon 35 had gene polymorphisms，nucleotide mutated from T to A，amino acids mutated from Leu to Glu，and the mutation rate reached 42.9%(9/21).All of the 29 ESBLs-producing stains were sensitive to imipenem. The resistance rates to cefoxitin,cefepime and ceftazidime were 29.4%，11.8% and 41.2% for Escherichia coli and 50.0%，8.3% and 33.3% for Klebsiella pneumoniae,respectively. More than 75% of the stains were highly resistant to ampicillin，piperacillin，cefazolin，cefuroxime and cotrimoxazole，while all of them were resistant to other drugs with variable degrees.  Conclusions　Pyrosequencing can be used to rapidly detect resistant genotyping of clinical isolated ESBLs-producing strains with advantages of accuracy，speediness，real-time implementation and high-throughput.

Key words: SHV genotype, Pyrosequencing, Extended-spectrum beta-lactamase , Single nucleotide polymorphism