Diatom Fingerprints: Multiplex PCR-HRM Offers a Rapid New Way to Identify Mixed Diatom Species

Diatoms, the microscopic single-celled algae that drift through nearly every river, lake, and ocean on Earth, are now at the center of a technological breakthrough that could transform both environmental science and forensic medicine. These tiny organisms, encased in ornate silica shells, are not only the engines of the global carbon cycle and a bellwether of aquatic ecosystem health but also silent witnesses in drowning investigations. When a person drowns, diatoms enter the lungs through respiration and spread via the bloodstream to organs such as the liver and kidneys. Detecting them in these tissues strongly supports a diagnosis of drowning, and identifying their species can help forensic scientists infer where the drowning occurred. Yet identifying diatoms has always been a painstaking task, one that has traditionally relied on expert taxonomists peering through light or electron microscopes at thousands of specimens.

Now, a team of researchers led by Yunchao Zhou, Yiwei Liu, Bin Cong, Jianhua Chen, and Jianqiang Deng has published an experimental study in the journal iScience demonstrating that a combination of multiplex polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis can rapidly and accurately identify both single and mixed diatom species. The work, which screened 83 primer pairs and validated 15 for diatom detection, offers a compelling alternative to both traditional microscopy and expensive high-throughput sequencing, particularly for laboratories that lack access to costly sequencing infrastructure.

The study focused on eight species of pure marine diatoms: Navicula sp., Odontella sp., Nitzschia sp., Chaetoceros sp., Ditylum sp., Skeletonema sp., Cylindrotheca sp., and Asterionellopsis sp. Scanning electron microscopy confirmed that all eight cultured strains exhibited morphological characteristics consistent with their purchased taxa, ensuring that the molecular results could be confidently attributed to the correct organisms. The researchers then extracted DNA from each species and set about designing and screening primers targeting a range of genetic markers, including the 18S and 28S ribosomal RNA genes, the chloroplast genes rbcL and PsaA, and the universal plastid amplicon UPA.

Through repeated experiments, the team identified 15 primer pairs capable of diatom detection—eight drawn from the published literature and seven designed in-house. When each primer pair was used to amplify DNA from a single diatom species followed by HRM analysis, the results were striking. Each amplification product formed a distinct characteristic peak in the derivative melting curve, with the horizontal coordinate of the highest point corresponding to the melting temperature. Different species yielded peaks of varying shapes at distinct positions, enabling identification based on these features alone. Nine primers—UPA99, 18S-V7, PsaA-2, rbcL197, DiatrbcL708, Primer1, ND, 28S-191C, and 18S-333N—could specifically amplify all eight species, each producing eight distinct melting curves. Others were more selective: 18S-356H and 18S-341J amplified seven species, rbcL-256Z amplified four, 18S-196O and rbcL-N amplified only two, and DNGT amplified only Skeletonema sp. No detection peaks were observed in any negative control group, underscoring the specificity of the approach.

However, the real challenge lay in mixed samples. In natural water environments, diatoms almost never appear as pure cultures; they exist as complex communities of many species. When the researchers used a single primer pair to amplify pairwise mixtures of diatoms, they observed a troubling phenomenon: mutual inhibition during amplification or fusion of characteristic peaks among species with similar melting temperatures. For example, when Navicula sp. was mixed with six other species using the DiatrbcL708 primer, only the characteristic peaks of the other diatoms appeared, without forming double peaks with Navicula sp. Asterionellopsis sp. proved particularly susceptible to suppression in mixed systems, its signal vanishing entirely when combined with most other species. When the researchers attempted to amplify a mixture of all eight species with a single primer, the resulting curves formed only double peaks that could not be attributed to any specific pair of species, demonstrating that single-primer HRM analysis lacks the discriminatory power needed for complex mixed samples.

The solution emerged from combining multiple primer pairs within a single reaction tube. The researchers first demonstrated that a mixture of two primers with similar annealing temperatures—28S-191C and PsaA-2—could form characteristic double peaks when amplifying a single diatom, with each peak corresponding to the signal produced by the respective primer alone. No amplification inhibition or peak fusion occurred, confirming that multiple primers can coexist in one reaction without interfering with each other. Building on this principle, the team then combined the primers 18S-196O, rbcL-N, and DNGT into a multiplex PCR-HRM system. This three-primer combination successfully distinguished four diatom species—Odontella sp., Navicula sp., Cylindrotheca sp., and Skeletonema sp.—in mixed samples. Remarkably, the melting curves produced by the mixed samples of four species and the mixed samples of all eight species were almost identical in shape and position, indicating that the system could simultaneously demonstrate the presence of these four species within a complex mixture.

The success of the multiplex PCR-HRM approach hinges on careful primer design. The annealing temperatures of primers must not differ by more than 4°C, or they cannot react efficiently in the same PCR system. At the same time, the melting temperatures of the amplified products must not be too close, or fusion peaks will form and species cannot be distinguished. The researchers noted that when the length of an amplified product exceeds 400 base pairs, the sensitivity and specificity of HRM analysis decline slightly. The most common problem encountered during the experiments was complementarity between primers, which produced excessive primer dimers. Fortunately, during HRM analysis, the presence of primer dimers can be observed directly in the melting curves, eliminating the need for agarose gel electrophoresis. Other factors influencing the system include the ratio of primers, the concentration of deoxynucleotide triphosphates (dNTPs), and the concentration of magnesium ions. Excessively high primer concentrations lead to competition among primers for PCR materials, causing amplification imbalance. Insufficient dNTP concentrations reduce the yield of amplification products, while inadequate magnesium ion concentrations impair polymerase activity, reducing amplification efficiency. Conversely, excessively high magnesium ion concentrations increase nonspecific amplification.

When the researchers performed HRM analysis on the eight diatom species using a single primer, they found that the melting temperatures of amplification products from different species were sometimes identical or differed by less than 0.1°C. For instance, the melting temperature of both Chaetoceros sp. and Cylindrotheca sp. amplified by the UPA99 primer was 79.28°C. This could be due to the similarity of the amplified sequences or to fluctuations in temperature differences between wells on the real-time PCR instrument. The researchers addressed this ambiguity by mixing two primers in the same reaction tube to amplify the diatoms, and if two primers still could not identify a species, a combination of three primers could be employed. However, the more primers used, the more difficult the experiment becomes to optimize and interpret.

Compared to high-throughput sequencing, which remains the most effective molecular approach for identifying species in complex mixed samples, multiplex PCR-HRM offers distinct practical advantages. Sequencing can simultaneously obtain DNA sequence data from a large number of species and has the potential to reveal unknown species within complex samples, but it is limited by high costs, long experimental cycles, and vast and complex data analysis requirements. It also depends heavily on the completeness and reliability of reference databases. Multiplex PCR-HRM, by contrast, completes PCR amplification and HRM detection in the same closed tube without any intermediate operations, reducing the risk of cross-contamination while simplifying the process and saving time. The technology requires only a high-resolution real-time fluorescence PCR instrument and corresponding reagents, resulting in relatively low costs that place it within reach of basic laboratories.

The researchers acknowledge that their current system can detect only four types of diatoms from a mixture of eight samples, a limitation they attribute to an insufficient number of specific primers and differences in amplification efficiency among them. They plan to continue designing primers for additional diatom species and to optimize the multiplex PCR-HRM system by adjusting the PCR reaction conditions, verifying the sensitivity of each primer, and refining the concentration ratios between primers. Importantly, the study did not validate the system using environmental water samples, so its performance on unknown samples remains to be determined. Nevertheless, this preliminary demonstration establishes the feasibility of the multiplex PCR-HRM approach for analyzing mixed diatom samples, offering a rapid, specific, low-cost, and operationally simple method with promising prospects for both environmental monitoring and forensic practice. As climate change and pollution continue to pressure aquatic ecosystems worldwide, and as forensic laboratories seek faster and more reliable tools for drowning diagnosis, this elegant fusion of molecular biology and thermal analysis may soon find itself at the forefront of diatom identification technology.

Subject of Research: Molecular Diagnostics

Article Title: An experimental study on HRM combined with multiplex PCR for the identification of single and mixed diatom species

Article References: Zhou, Y., Liu, Y., Cong, B., Chen, J., & Deng, J. (2026). An experimental study on HRM combined with multiplex PCR for the identification of single and mixed diatom species. iScience, 29(11), Article 117909. https://doi.org/10.1016/j.isci.2026.117909

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117909

Keywords: experimental, combined, multiplex, identification, single, mixed, diatom, species, scientific research