title: Handheld device for real-time, quantitative, LAMP-based detection of Salmonella enterica using assimilating probes.
abstract: A simple handheld instrument was designed to enable real-time detection of the LAMP reaction in a standard PCR tube using newly described assimilating probes as sequence-specific reporter molecules. The system was validated using DNA isolated from Salmonella enterica, demonstrating accurate temperature control with little power and little overshoot of setpoint temperatures, with rapid and accurate detection often in less than 30 min and within 20 min for reactions with high (>10(5)) genome copy numbers. The system could be used for quantitative determination of pathogen DNA, with a limit of detection of about 15 genome copies in purified DNA or 25 cells in DNA extracts from chicken rinsate--comparable to values obtained when running the same reaction on a commercial benchtop real-time PCR instrument. Positive classification of standards nominally containing a single genome equivalent was demonstrated, and no false positives were reported. Detection of S. enterica in rinsate from a contaminated chicken sample required 48 h enrichment prior to the LAMP reaction or plating on semi-selective media. The new system demonstrates a major compelling advantage of the LAMP reaction, in that it may be enabled in simple, low-power, handheld devices without sophisticated custom miniaturized disposables. This new diagnostic system is especially promising for on-site diagnostics in the food and agricultural industries where laboratory space is often primitive if it is available at all.
authors: Department of Molecular Biosciences and Bioengineering, University of Hawai'i, Mānoa, USA. danielje@hawaii.edu
authors: Daniel M
authors: DM
authors: Jenkins
authors: Ryo
authors: R
authors: Kubota
authors: Jin
authors: J
authors: Dong
authors: Yong
authors: Y
authors: Li
authors: Dennis
authors: D
authors: Higashiguchi
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005504:Food Analysis / Q000295:instrumentation*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D008904:Miniaturization; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial