High-Resolution Melting Analysis Method for Sensitive Detection of Plant Pathogens
Authors: A. Kandan, J. Akhtar, Pardeep Kumar and Z. Khan
Division of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi - 110012


Sensitive techniques are urgently needed so that a pathogen can be detected at an early stage of host infection to avoid the yield loss, eventually economic loss to the farmers. High-Resolution Melting Analysis (HRMA) is relatively a novel technique which is very specific and sensitive in pathogen detection, genotype scanning and rapidly identifies DNA sequence variants, without needing cumbersome post-PCR techniques. HRMA is high throughput technique to identify fungi and bacteria in planta even when they are present in very small amounts.

Principles of HRMA

For detection of plant pathogens/food pathogens from apparently infected plant or food samples, post-PCR melt curve analysis using SYBR® Green I dsDNA intercalating dye was used to detect either primer-dimers or other non-specific products which is so called as Low Resolution Melting (LRM). LRM curve is produced by increasing the temperature gradually, as typically in 0.5°C increments, which lead gradual denaturing of an amplified DNA target. As SYBR® Green I is only fluorescent oriented when bound to dsDNA, fluorescence decreases as duplex DNA is denatured under high temperature. Always the melting profile depends on the length, genomic sequence, GC content, and heterozygosity of the amplified target. The main principle of HRMA is the same as a LRM, except that the temperature difference between each fluorescence reading is reduced as the temperature are increasing phase. During a LRM curve analysis, the temperature increases are typically in 0.5°C steps, but for HRMA this is reduced to 0.008 - 0.2°C increments. This difference in temperature increments allows a much more detailed analysis of the melting behavior. HRMA sensitivity and reliability has been improved with the use of a variety of new dsDNA intercalating dye mainly Evagreen.

The first step of the HRMA protocol is the amplification of the specific region of interest, using standard PCR techniques, mainly in the presence of a specialized double-stranded DNA (dsDNA) binding dye. This specialized evagreen dye is highly fluorescent when bound to dsDNA and poorly fluorescent in the unbound state of the targeted genomic region. This effective change allows the scientists to monitor the DNA amplification during PCR (as in quantitative PCR). After successful completion of the PCR step, the amplified target is gradually denatured by increasing the temperature in small increments, in order to produce a characteristic melting profile which is termed as a melting analysis. The amplified target denatures gradually, releasing the dye, which results in a drop in fluorescence is recorded and analyzed using software.

Applications of HRMA

HRMA very clearly exploits the fact that targeted PCR products with different sequences have very distinct melting profiles. At high melting temperature, the signal change that signifies the transition from a double to a single strand is generated by fluorescent dyes that actively intercalate double stranded DNA with very low interference with the PCR reaction. In this HRMA reaction, a third-generation intercalating dyes such as EvaGreen have been successfully used for HRMA on various real-time cyclers. These type of dyes have low levels of inhibition and can therefore be used at higher concentrations for greater saturation of the dsDNA sample. In HRMA reaction, greater dye saturation means that the fluorescent signals measured have higher fidelity, and because there is less dye redistribution to nondenatured regions of the DNA strand during melting, and because dyes do not favor products with a higher melting temperature profile. The combination of these specific characteristics provides greater melt sensitivity and higher resolution melt profiles, which resulting in more distinct melting curves. The HRMA profile of a PCR product produces a sequence-related curve, specific that rapidly distinguishes between sequences even when they differ by only one nucleotide, as seen in complex applications such as SNP detection, microbial typing, as well as mutation analysis.

Advantages of HRMA

1. Requirement of very low reagent for detection of plant pathogen or SNP genotyping.

2. It is simple and fast workflow. There is no additional instrumentation is required after PCR amplification. A HRMA curve can be added to the end of the amplification run and analyzed immediately after completion of PCR.

3. Fast optimization is possible.

4. Low sample consumption as after HRM analysis, the PCR amplicon can be used directly in a Sanger sequencing reaction.

As the HRMA technique meets all the conditions required for early detection of plant pathogens, it is a rapid, close-tube, highly efficient and low-cost post-PCR approach that permits a rapid screening of the pathogen before any symptoms appear in the hosts. For this specific reason HRMS could also be used to evaluate the effectiveness of treatments against different plant pathogens, and also acts as a effective tool to examine planting stock or seeds for quarantine purposes.

HRMA promote very high-throughput screenings of host which are symptomatic or asymptomatic which aimed at revealing important epidemiological data and trends of this species, such as temporal or geographical tendencies, host range, and other fundamental observations related to pathogenicity and/or virulence of the pathogens. In addition the intrinsic high relevance of these epidemiogical findings of any phytopathogens, HRMA data would also strongly influence the management of the diseases caused by the pathogens by helping to correctly calibrate preventive disease control measures concerning these phytopathogens. Since HRMA can be performed in an economical, rapid and convenient way, it could be easily applied in routine identification of phytopathogens in seed and plant propagation materials by a simple high-throughput process.

About Author / Additional Info:
Senior Scientist (Plant Pathology), Division of Plant Quarantine, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110012.