title: Heptaplex PCR melting curve analysis for rapid detection of plasmid-mediated AmpC β-lactamase genes.
abstract: Antimicrobial resistance mediated by plasmid-borne AmpC β-lactamase in Gram-negative bacteria is an emerging event of significant clinical importance. Rapid and reliable detection of ampC is in urgent need for appropriate infection control. We described the development and evaluation of a heptaplex PCR melting curve analysis that could identify six groups of ampC, i.e., CIT, EBC, DHA, ACC, MOX and FOX, through predefined melting temperatures. The entire analysis could be finished within 2h for 96 samples after template DNA was prepared. We first evaluated the assay with 176 AmpC-producing isolates of Escherichia coli and Klebsiella pneumoniae, and the results showed that 36 isolates were positive for ampC, including 18 positive for DHA, 12 for CIT, 5 for EBC, and one for both DHA and EBC. These results were fully concordant with sequencing analysis whereas the comparison method, an electrophoresis-based singleplex PCR assay, missed four isolates. The assay was also used to analyze 429 randomly selected clinically relevant Gram-negative isolates involving 22 different species, and 34 isolates were found to be ampC-positive. The results again fully agreed with the sequencing analysis. We conclude that the established assay could be used for rapid and reliable detection of ampC.
authors.affiliation: The State Key Laboratory of Cellular Stress Biology; State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; Engineering Research Center of Molecular Diagnostics, Ministry of Education; School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Zanzan
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, China.
authors.forename: Jiaqin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Cellular Stress Biology; State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; Engineering Research Center of Molecular Diagnostics, Ministry of Education; School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Shitao
authors.initials: S
authors.lastname: Rao
authors.affiliation: The State Key Laboratory of Cellular Stress Biology; State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; Engineering Research Center of Molecular Diagnostics, Ministry of Education; School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.affiliation: The State Key Laboratory of Cellular Stress Biology; State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; Engineering Research Center of Molecular Diagnostics, Ministry of Education; School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Cellular Stress Biology; State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; Engineering Research Center of Molecular Diagnostics, Ministry of Education; School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, China.
authors.forename: Gangsen
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, China.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, China.
authors.forename: Shuyi
authors.initials: S
authors.lastname: Hou
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, China.
authors.forename: Xinguo
authors.initials: X
authors.lastname: Zhuang
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, China. Electronic address: songxyxm@hotmail.com.
authors.forename: Xiuyu
authors.initials: X
authors.lastname: Song
authors.affiliation: The State Key Laboratory of Cellular Stress Biology; State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics; Engineering Research Center of Molecular Diagnostics, Ministry of Education; School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China. Electronic address: qgli@xmu.edu.cn.
authors.forename: Qingge
authors.initials: Q
authors.lastname: Li
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010957:Plasmids; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D047090:beta-Lactams; C048795:AmpC beta-lactamases; D001618:beta-Lactamases
keywords: Gram-negative bacterium; Melting curve analysis; Multiplex PCR; Plasmid-mediated AmpC β-lactamase