title: Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B.
abstract: We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries.
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Cuiming
authors.initials: C
authors.lastname: Cao
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Mingyu
authors.initials: M
authors.lastname: Ji
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Fengyan
authors.initials: F
authors.lastname: Pei
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Xiujie
authors.initials: X
authors.lastname: Fan
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Shen
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China.
authors.forename: Qingxi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China. Electronic address: whyxp@163.com.
authors.forename: Weihua
authors.initials: W
authors.lastname: Yang
authors.affiliation: Medical Research & Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong, 250013, China. Electronic address: sdjnwys@163.com.
authors.forename: Yunshan
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D021141:Nucleic Acid Amplification Techniques; D012680:Sensitivity and Specificity; D013297:Streptococcus pyogenes / Q000235:genetics*
chemical_list: D001427:Bacterial Toxins; D003546:Cysteine Endopeptidases; C042477:streptopain
keywords: Loop-mediated isothermal amplification (LAMP); Primers; Rapid detection; Streptococcus pyogenes; speB gene