title: Detection of Salmonella in chicken meat by insulated isothermal PCR.
abstract: Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10⁰ CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.
authors.affiliation: Department of Food Science and Technology, Hung Kuang University, No. 1018, Sec. 6, Taiwan Boulevard, Shalu District, Taichung City 43302, Taiwan, Republic of China.
authors.forename: Hau-Yang
authors.initials: HY
authors.lastname: Tsen
authors.forename: Chia-Ming
authors.initials: CM
authors.lastname: Shih
authors.forename: Ping-Hua
authors.initials: PH
authors.lastname: Teng
authors.forename: Hsin-Yen
authors.initials: HY
authors.lastname: Chen
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Lin
authors.forename: Chien-Shun
authors.initials: CS
authors.lastname: Chiou
authors.forename: Hwa-Tang Thomas
authors.initials: HT
authors.lastname: Wang
authors.forename: Hsiao-Fen Grace
authors.initials: HF
authors.lastname: Chang
authors.forename: Te-Yu
authors.initials: TY
authors.lastname: Chung
authors.forename: Pei-Yu
authors.initials: PY
authors.lastname: Lee
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Chiang
mesh_terms: D000818:Animals; D002645:Chickens / Q000382:microbiology*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005452:Fluoresceins; D005506:Food Contamination / Q000032:analysis*; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012235:Rhodamines; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012478:Salmonella Food Poisoning / Q000517:prevention & control; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: C106141:6-carboxytetramethylrhodamine; D017931:DNA Primers; D004269:DNA, Bacterial; D005452:Fluoresceins; D012235:Rhodamines; C024098:6-carboxyfluorescein