Evaluation of DNA melting analysis as a tool for species identification

Abstract

Summary 1.One of the ongoing challenges in molecular biology is to develop rapid, accurate and reliable techniques to identify organisms. This contribution evaluates the value of DNA melt peak analysis for the identification of pests and pathogens significant to biosecurity. 2. The method evaluated in this study capitalises on the observation that double-stranded DNA separates into single strands (melts) at a temperature dependant on the nucleotide sequence of the DNA strands. The temperature and shape of melt peaks from polymerase chain reaction (PCR) products using standard primers and amplification protocols were evaluated for species identification, and the advantages of this method over agarose gel electrophoresis exemplified. 3. Three insect (weevil) species were discriminated by the melt profile shape and temperature of their cytochrome oxidase subunit I (COI) PCR product using order-specific universal primers. 4. Three of four arachnid (tick) species were discriminated by the melt profile shape and temperature of their COI and 18S PCR product using order-specific universal primers. 5. Plum pox virus-specific primers amplified only the target virus that was verified by its unique melt temperature rather than by conventional staining of the amplicon on an electrophoretic gel. 6. Melt peak analysis is a useful and robust tool for the rapid detection of PCR products and the identification of organisms. An efficiency study revealed this technique required half the time input, and cost half as much, as PCR followed by AGE.

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

“…The present work describes a primer pair, SalF and SalR, based on the 16S rRNA gene and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis as a sensitive, specific and inexpensive method for the identification and quantification of the fish pathogen Vagococcus salmoninarum in bacterial cultures and infected fish tissue homogenates. This qPCR diagnostic‐based method can robustly monitor and quantify amplified products in a single tube in “real time” without the need for electrophoresis gels (Winder et al, ), avoiding post‐processing steps and delays, and minimizing potential experimental error. This assay can be completed in <3 hr, including DNA preparation from bacterial cultures or fish tissues, qPCR amplification and melting curve analysis.…”

“…In SYBR Green qPCR assays, the amount of PCR products (amplicons) can be detected by measuring fluorescence levels from the cycle threshold ( C q ) (Winder et al, ). Therefore, based on the observed values of C q and the linear standard curve generated, the SalF and SalR primers designed in this study showed the highest sensitivity and were estimated to have a detection limit of 0.034 × 10 0 copies of amplicon by μl (equivalent to 2.0 × 10 −11 ng/µl genomic DNA).…”

“…Real‐time or quantitative PCR (qPCR) is a technique that has been widely used for the detection and, simultaneously, the quantification of pathogens responsible for streptococcosis in fish such as Lactococcus garvieae (Jung, Chang, & Kim, ), Streptococcus parauberis (Nguyen, Lim, Kim, & Austin, ) or Streptococcus agalactiae (Sebastião Fde, Lemos, & Pilarski, ) using either a fluorescent dye that intercalates with double‐stranded DNA or a modified DNA oligonucleotide (DNA probe) that fluoresces when hybridized with complementary DNA. Real‐time PCR with the intercalating dye SYBR Green I combined with melting curve analysis is a reliable inexpensive technique that allows highly specific and sensitive detection and analyses of PCR products in a single tube in “real time,” which decreases post‐processing steps and delays, and minimizes potential experimental error (Winder et al, ). This combinatorial technique has been used for the detection and identification of other fish pathogens such as Aeromonas salmonicida subspecies salmonicida (Fernández‐Álvarez, González, & Santos, ), Tenacibaculum maritimum (Fernández‐Álvarez, González, & Santos, ) or Photobacterium damselae subspecies piscicida (Carraro et al, ).…”

Identification of the GST-T1 and GST-M1 Null Genotypes Using High Resolution Melting Analysis

“…However, it may not work well for the HRM analysis in situations when short PCR fragments are amplified as it results in a weak fluorescence emission, yielding small, hard to detect melting peaks. 30-35 Thus, “saturation dyes” such as LC Green Plus, SYTO9, Eva Green, or ResoLight have been developed to be used with the HRM technique. 33,36 These dyes do not inhibit PCR amplification even at concentrations that completely saturate DNA.…”

Species detection using HyBeacon ® probe technology: Working towards rapid onsite testing in non-human forensic and food authentication applications

“…They tend to have high sensitivity due to the use of polymerase chain reaction (PCR), high specificity to the chosen target and can be used on highly processed samples, many of which have been exposed to high temperatures [9]. Common DNA techniques using PCR include PCR-restriction fragment length polymorphisms (PCR-RFLP), amplified fragment length polymorphisms (AFLP), forensically informative nucleotide sequencing (FINS), random amplified polymorphic DNA (RAPD), melt curve analyses [10,11] and DNA sequencing [12]. A lack of governance and standardisation relating to species identification in food standards means that each laboratory often develops ad-hoc approaches, many of which have been phased out of use in routine forensic applications.…”