Improvements to the Rapid Detection of the Marine Pathogenic Bacterium, Vibrio harveyi, Using Loop-Mediated Isothermal Amplification (LAMP) in Combination with SYBR Green

Abstract

The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi. The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non-Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field.

Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2

“…Six primers were designed for each set of primers, a pair for the inner and outer primers, respectively, as well as a pair of loop primers. Loop primers were found to increase the amplification speed of LAMP [ 19 , 30 , 43 ] as well as increase its specificity [ 44 ].…”

“…The target N, M and E genic regions were amplified via PCR using the outer primers of each primer set. The PCR reactions were as described [ 30 ] with modifications. The reaction of 25 µL consisted of a 1X PCR buffer, 1.5 mM MgCl 2 , 0.2 mM dNTP mix (Promega, Madison, WI, USA), 20 pmol of each outer primer and 0.2 U Taq DNA polymerase (Promega, Madison, WI, USA).…”

“…The recombinant plasmid DNA with each gene was serially diluted 10-fold to achieve 10 8 copies to one copy number [ 30 ]. Both the endpoint and quantitative PCR were conducted on each dilution of recombinant plasmid using the outer primers stated in Table 1 .…”

“…The 1.5% agarose gel electrophoresis was conducted on amplification products, stained in ethidium bromide and observed under UV conditions. Colorimetry using SYBR Green was conducted by the addition of 2 µL of 1:10, which was diluted SYBR Green I nucleic acid gel stain to all tubes containing LAMP products, and observations on the color changes were immediate [ 30 ]. As for LFD, the LAMP protocol was conducted using the primers stated in Table 1 , where inner primers were labeled with specific antigens for LFD detection.…”

Loop-mediated Isothermal Amplification (LAMP) assay for reliable detection ofXanthomonas axonopodispv.vasculorum

“…Results of LAMP can be verified by electrophoresis, precipitation of magnesium pyrophosphate byproduct, or SYBR green assay 17,24 . SYBR green assays use a fluorescent intercalating dye present in the solution that binds to double-stranded DNA and can be confirmed visually with the naked eye or under UV light 18 .…”