Detection of Bacteria Carrying the stx ₂ Gene by In Situ Loop-Mediated Isothermal Amplification

Abstract

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA 2 in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA 2 gene. Higher-contrast images were obtained with this method than with in situ PCR.

Methods for Whole Cell Detection of Microorganisms

“…Over the past decade, several distinct methods for generating sequence-specific fluorescence signals within intact bacterial cells have been developed, primarily for environmental microbiology applications. These include in situ PCR (25)(26)(27)(28)(29), in situ reverse transcription-PCR (30)(31)(32), in situ reverse transcription (33), chromosomal painting (34,35), in situ rolling circle amplification (36,37), in situ loop-mediated signal amplification (38) and fluorescence in situ hybridization (FISH) (39)(40)(41)(42)(43). Descriptions of each method are given below, followed by a discussion of their potential benefits and drawbacks for use in routine diagnostics in environmental, food and clinical microbiology applications.…”

“…As originally described, loop-mediated isothermal amplification of DNA (LAMP) is a method capable of quickly amplifying select DNA sequences, enabling production of up to 10 9 copies within one hour (48). The LAMP procedure relies on a self-sustaining strand displacement reaction carried out by a DNA polymerase having high strand displacement activity, in this case the Bst large fragment from Bacillus stearothermophilus (38,48). The technique uses up to six distinct primers that hybridize with the target sequence to create the topologically unique nucleic acid structures used for amplification (38,48).…”

“…The LAMP procedure relies on a self-sustaining strand displacement reaction carried out by a DNA polymerase having high strand displacement activity, in this case the Bst large fragment from Bacillus stearothermophilus (38,48). The technique uses up to six distinct primers that hybridize with the target sequence to create the topologically unique nucleic acid structures used for amplification (38,48). As adapted for in situ detection of target genes within bacterial cells, only four LAMP primers were required and specific amplification products could be detected either via FISH with ampliconspecific probes, or via incorporation of fluorescently-labeled dCTP during amplicon formation (38).…”

“…The technique uses up to six distinct primers that hybridize with the target sequence to create the topologically unique nucleic acid structures used for amplification (38,48). As adapted for in situ detection of target genes within bacterial cells, only four LAMP primers were required and specific amplification products could be detected either via FISH with ampliconspecific probes, or via incorporation of fluorescently-labeled dCTP during amplicon formation (38). As with./?!…”

“…As with./?! situ RCA, in situ LAMP has no requirement for complicated, expensive cycling equipment and the relatively low temperature used (63°C) is potentially less destructive to delicate cell structures, preserving cell morphology and diagnostic epitopes (38).…”