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.

Keywords: LAMP-SYBR green; Vibrio harveyi; loop-mediated isothermal amplification; rapid detection; toxR gene; vibriosis.

Figure 1

The flanking regions of the inner (green), outer (yellow) and loop (blue) primers based on the toxR gene of V. harveyi for LAMP reaction.

Figure 2

Gel electrophoresis of the LAMP assay and visualization with SYBR Green (A) An optimized LAMP protocol was developed for subsequent downstream tests, lane 1: V. harveyi (genomic DNA), lane 2: 100 bp DNA ladder, lane 3: negative control (distilled water). (B) Reaction mixture without loop primers, lane M: 100 bp DNA ladder, lane 5: negative control (distilled water); the reaction time of LAMP is indicated on the top of each lane. (C) Reaction mixture with loop primers, lane M: 100 bp DNA ladder, lane 1: negative control (distilled water); the reaction time of LAMP is indicated on the top of each lane. All products were analyzed by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. (D) Visual appearance of LAMP coupled with SYBR Green. Positive sample (green) and negative sample (orange).

Figure 3

Results of sensitivity tests using PCR, LAMP-UV and LAMP-SYBR Green using different concentrations of recombinant plasmid of V. harveyi from 60 ng to 0.6 fg. End-point PCR (A, top), LAMP-UV analysis (A, bottom) and LAMP-SYBR Green (B). The concentrations of samples are indicated on the top of each lane and tube. Lane M: 100 bp DNA ladder, Lane 10: negative control (distilled water).

Figure 4

Specificity test for LAMP-SYBR green indicating the presence of amplicons for only V. harveyi. (A) LAMP amplicons visualized with gel electrophoresis in a 1.5% agarose gel and stained with ethidium bromide. (B) The corresponding LAMP assay visualized with SYBR green. Lane 1: 100 bp DNA ladder, Lanes 2–3: negative controls (distilled water), Lane 4: Aeromonas salmonicides subsp. salmonicida ATCC strain 33658, Lane 5: V. aguillarium ATCC 19264, Lane 6: V. alginolyticus ATCC 17749, Lane 7: V. parahaemolyticus ATCC 17802, Lane 8: V. harveyi strain VHJR 7.

Figure 5

Evaluation of the improved LAMP-SYBR Green protocol on Vibrio harveyi infected grouper. (A) Sample of infected fish. (B) Sample of control non-infected fish. Tissue samples were obtained after 48 hrs of treatment, and the DNA template was prepared using the rapid-boiling method. The corresponding LAMP assay showed as being positive (green) for all three replicates, while the controls were negative (orange).