Pearl River Estuary field study finds multiplex PCR sequencing detects targeted aquatic invasive species

A field study combining environmental DNA, multiplex PCR and amplicon sequencing detected 28 aquatic invasive species across 32 sites in China’s Pearl River Estuary. The assay, designed for targeted screening of known invasive taxa, achieved a 90% detection rate when validated against mixed DNA standards for 46 species, although five species failed to amplify in the multiplex system.

The researchers selected 46 harmful aquatic invasive species from China using literature, official documents and open databases. They prepared 91 species-specific primer pairs: 31 pairs adapted from earlier studies for 20 species and 60 newly designed pairs for 31 species using mitochondrial sequences. The new primers primarily targeted the mitochondrial COI and D-loop regions. Each pair successfully amplified DNA from its corresponding tissue when tested individually.

Validation of the combined assay produced a different result from those individual tests. When all 91 primer pairs were used with mixed standard plasmids in one multiplex reaction, 70 amplified their target sequences. Those primers detected 41 of the 46 species, corresponding to a species detection rate of 90%. Five species—apple snail, alligator snapping turtle, rainbow trout, Mozambique tilapia and common snapping turtle—did not amplify. The researchers attributed the failures to possible interactions among primers, which require annealing temperatures 4–6°C lower than single-target PCR, and to possible competition from abundant non-target DNA.

For environmental testing, the team collected three 1-litre water samples at each of 32 sites in July 2021, although 40 sites had initially been planned. Each sample was filtered through three mixed-cellulose membranes with 0.45-micrometre pores. DNA was extracted from the membranes, and a 500-millilitre filtered-water sample served as a negative control during sampling, extraction and PCR. The environmental multiplex reactions used a 30-microlitre mixture containing 15 microlitres of master mix, 6 microlitres of primer mixture, 8 microlitres of water and 1 microlitre of DNA template.

In the field assay, 33 primer pairs amplified targets and detected 28 aquatic invasive species. Five fish species were each detected with two primer pairs. Amphibalanus amphitrite, a marine barnacle that can attach to ship hulls and buoys, had the highest relative sequence abundance, followed by tench, Coptodon zillii and Nile tilapia. The relative abundance of A. amphitrite was especially high at estuarine sites such as L26, L27, L37 and L40, while Nile tilapia had a significantly dominant relative abundance at inland sites such as L3, L13 and L34.

Several detected species were recorded at all 32 sampling sites, including red-bellied piranha, coho salmon, Nile tilapia, rohu and red drum. Six sites—L2, L3, L10, L31, L38 and L40—each contained more than 22 detected invasive species. The patterns were consistent with ecological and transport factors discussed by the authors: tilapia were associated mainly with inland waters, whereas the tendency of A. amphitrite to attach to vessels and buoys may facilitate movement between estuarine and inland sites. These associations did not establish the causes of the observed distributions.

The study compared the targeted multiplex assay with fish metabarcoding using universal 12S rRNA primers. Metabarcoding detected 91 fish species, including 11 invasive fish, and all 11 were also detected by multiplex PCR. Across those 11 co-detected species, Pearson analysis found significant correlations for seven: five were classified as highly significant at p<0.01 and two as significant at p<0.05. C. zillii and Nile tilapia had R² values above 0.75. Four species did not show significant species-level correlations, and low-abundance detections differed between methods. For example, Pterygoplichthys pardalis was clearly detected by 12S metabarcoding at sites L3 and L10 but was rarely observed through multiplex PCR.

Multiplex PCR detected 17 additional invasive species that were not covered by the fish-focused 12S assay, including crustaceans and molluscs. Its reported cost was comparable to metabarcoding, lower than digital PCR and higher than quantitative PCR. The authors position the two approaches as complementary: metabarcoding is suited to broad fish-community surveys, whereas multiplex PCR provides targeted screening across fish and non-fish taxa with a relatively low sample-volume requirement. These findings come from one estuarine field application and do not establish performance for every invasive species or monitoring environment.

Several limitations constrain the comparison. The 12S primers were designed primarily for fish and therefore were not expected to cover all non-fish invasive taxa, limiting a direct assessment of those groups. The multiplex assay also did not detect every target included in the standard-plasmid validation, and the authors note that results may be less reliable when sequence counts are low. In addition, the absence of species such as the common snapping turtle from estuarine detections may reflect low population density in the sampled area rather than absence throughout the wider Pearl River Basin. The study therefore supports further evaluation of primer concentrations or split-pool amplification for targets that fail under combined conditions.

The monitored distribution also provides context for management priorities. Tilapia were mainly introduced for aquaculture and have spread through human-mediated dispersal and movement within river systems. Previously reported enclosure experiments found that Nile tilapia could increase nutrient loading and phytoplankton biomass while reducing water transparency. Based on the present sequence data, the authors recommend prioritising ecological interception in high-abundance areas, citing blue tilapia’s 44% relative abundance at site L34. The monitoring results identify locations for targeted management, but the study did not test whether such measures reduce invasion or ecological effects.

Subject of Research: Environmental-DNA multiplex PCR sequencing for targeted monitoring of aquatic invasive species in the Pearl River Estuary.

Article Title: Invasive species monitoring based on eDNA multiplex PCR sequencing

Article References: Liu, Z., Du, X., Zhang, Z., Mu, Y., Yang, J., Yang, J., & Zhang, X. (2025). Invasive species monitoring based on eDNA multiplex PCR sequencing. Metabarcoding and Metagenomics, 9, Article e159944. https://doi.org/10.3897/mbmg.9.159944

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.159944

Keywords: multiplex PCR, environmental DNA, invasive species, amplicon sequencing, metabarcoding, aquatic monitoring, Pearl River Estuary, China, species-specific primers, Invasive, species, monitoring