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<p>This study addressed the challenge of accurately identifying stranded marine mammal species which currently relies heavily on morphological characteristics and is prone to damage or incompleteness. A DNA barcode combined with high resolution melting analysis (Bar-HRM) was used for accurate identification at the species level. Mitochondrial DNA, specifically the D-loop and cytochrome b gene, was utilized for Bar-HRM to differentiate 13 marine mammal species (<i>n</i> = 26) commonly found in Thai waters. Results demonstrated that Bar-HRM, using specific primers (MMCytb2 and MMCytb3), produced distinct normalized melting curves for all species (100.00% success rate). In blind tests, Bar-HRM demonstrated high accuracy rates in species differentiation, reaching 86.67% and 90.00% for Bar-HRM using MMCytb2 and MMCytb3 primers, respectively. The method also enabled 100.00% species identification in unknown samples (<i>n</i> = 20), with a minimum DNA concentration requirement of 0.01 ng per reaction for both primers. Multiplex HRM incorporating MMCytb2 or MMCytb3 with the SRY gene for sex determination showed effectiveness rates (100.00%). Morever, species identification using Bar-HRM was more cost-effective than DNA barcode sequencing. Taken together, this approach showed considerable potential for improving marine mammal species identification with accuracy, speed and cost effectiveness, thus enhancing conservation efforts and aiding in the assessment of their endangered status.</p>

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Stranded carcass identification of marine mammal species by high resolution melting analysis using barcodes (Bar-HRM)

  • 期刊论文

  • 发表日期：

    2025年4月24日

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  • Anocha Poommouang,
  • Promporn Piboon,
  • Kongkiat Kittiwatanawong,
  • Phuping Sucharitakul,
  • Patcharaporn Kaewmong,
  • Korakot Nganvongpanit,
  • Kittisak Buddhachat

摘要

This study addressed the challenge of accurately identifying stranded marine mammal species which currently relies heavily on morphological characteristics and is prone to damage or incompleteness. A DNA barcode combined with high resolution melting analysis (Bar-HRM) was used for accurate identification at the species level. Mitochondrial DNA, specifically the D-loop and cytochrome b gene, was utilized for Bar-HRM to differentiate 13 marine mammal species (n = 26) commonly found in Thai waters. Results demonstrated that Bar-HRM, using specific primers (MMCytb2 and MMCytb3), produced distinct normalized melting curves for all species (100.00% success rate). In blind tests, Bar-HRM demonstrated high accuracy rates in species differentiation, reaching 86.67% and 90.00% for Bar-HRM using MMCytb2 and MMCytb3 primers, respectively. The method also enabled 100.00% species identification in unknown samples (n = 20), with a minimum DNA concentration requirement of 0.01 ng per reaction for both primers. Multiplex HRM incorporating MMCytb2 or MMCytb3 with the SRY gene for sex determination showed effectiveness rates (100.00%). Morever, species identification using Bar-HRM was more cost-effective than DNA barcode sequencing. Taken together, this approach showed considerable potential for improving marine mammal species identification with accuracy, speed and cost effectiveness, thus enhancing conservation efforts and aiding in the assessment of their endangered status.