title: Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella.
abstract: Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30 min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0 × 10(1)fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries.
authors.affiliation: The Technical Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd, Xinhua District, Shijiazhuang 050050, China.
authors.forename: Jianchang
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Food Science and Technology, Agricultural University of Hebei, No.38 Lingyusi Street, Baoding 071001, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: The Technical Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd, Xinhua District, Shijiazhuang 050050, China.
authors.forename: Lianxia
authors.initials: L
authors.lastname: Hu
authors.affiliation: The Technical Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd, Xinhua District, Shijiazhuang 050050, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Sun
authors.affiliation: The Technical Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd, Xinhua District, Shijiazhuang 050050, China.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: The Technical Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, 318 Heping West Rd, Xinhua District, Shijiazhuang 050050, China. Electronic address: drjingli2014@gmail.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002417:Cattle; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005453:Fluorescence; D005516:Food Microbiology / Q000379:methods*; D005517:Foodborne Diseases / Q000382:microbiology*; D006801:Humans; D057230:Limit of Detection; D008460:Meat / Q000382:microbiology*; D064307:Microbiota / Q000235:genetics; D008892:Milk / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D000067075:Vegetable Products / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C075518:invA protein, Bacteria
keywords: Food-borne disease; Microbiological testing; Real time PCR; Salmonella; Single primer isothermal amplification