Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick

Abstract

Prymnesium parvum is a toxic algal bloom (HAB)-forming species. The toxicity of this alga is a result of a collection of compounds known as prymnesins. Prymnesins exert harmful effects upon fish, shellfish, and mollusks, causing huge economic losses. In the present study, a new method was developed for the detection of P. parvum. The novel method utilizes isothermal amplification, known as recombinase polymerase amplification (RPA), in combination with lateral-flow dipstick (LFD). Herein, a set of primers and probes were designed for internal transcribed spacer (ITS) sequences, and a specific and sensitive RPA-LFD rapid detection method was established for P. parvum. Meanwhile, we verified its feasibility for the detection of environmental samples. It was demonstrated that the optimal amplification temperature and time for RPA were 39°C and 15 min. RPA/RPA-LFD was experimentally verified to be specific, demonstrating no cross-reaction with distinct control microalgae, and furthermore, the total time required for the RPA-LFD experiment was 20 min. Meanwhile, the detection limit for the genomic DNA of P. parvum was 1.5×10-1 pg/μL, and the detection limit for plasmids was 2.35 pg/μL. In addition, the results herein revealed that the RPA-LFD assay was 100 times more sensitive than PCR for detection of P. parvum. In conclusion, we developed an RPA-LFD that does not require precision instruments, and can be utilized for rapid on-site detection of P. parvum. In the future, the RPA-LFD can be considered for practical application for environmental detection of the toxic algal species.

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

“…To maximize the effectiveness of RPA-LFD detection, it is important to think about the parameters separately from the primers and probes. This includes the temperature and duration of the amplification process to increase the amplification effectiveness, shorten the process, save costs, and ensure that the RPA products produce a visible red band at the test line on the LFD strips [28]. All the aforementioned RPA-LFD systems were tested in this study at a temperature of 37 • C for 15 min, which required minimally complex instruments and fulfilled the stability and sensitivity requirements of in-field applications.…”

“…However, because of the method itself, it still has some defects. Such as RPA reactions produce more non-specific amplifications relative to PCR, a difficult phenomenon to avoid [28,51]. Therefore, it is necessary to screen primers in order to minimize the interference of non-specific amplification for subsequent experiments.…”

“…Due to the expertise and time that are required for the microscopic discrimination of species, molecular methods that monitor the environmental concentrations of Pseudonitzschia provide a rapid alternative for early bloom detection and toxin accumulation prediction. The main molecular detection methods for HAB species have included polymerase chain reaction (PCR) [18], quantitative PCR (qPCR) [19][20][21], enzyme-linked immunosorbent assay [22], rolling circle amplification [23], helicase-dependent amplification [24], nucleic acid sequence-based amplification [25], loop-mediated isothermal amplification (LAMP) [26], recombinase-aided amplification (RAA) [27], and recombinase polymerase amplification (RPA) [28], among others [29]. Currently, the detection of P. multiseries is mainly divided into two categories.…”

“…Furthermore, RPA's interoperability allows it to be widely utilized in conjunction with other product detection methods [38], such as agarose gel electrophoresis [37], real-time fluorescence probes [39], and lateral flow dipstick (LFD) [40]. When compared to other methods of detection, LFD's portability, ease of use, visualization, low cost, and quick setup time make it an attractive option [28]. In addition, DNA amplification using RPA and LFD readouts can be completed in under 30 min [41,42].…”

Review of Harmful Algal Blooms (HABs) Causing Marine Fish Kills: Toxicity and Mitigation

“…This mixotrophic haptophyte has been reported to orchestrate significant ecological effects, including enormous fish fatalities, in many environments because of its ichthyotoxicity. This organism is said to be mostly of marine origin but also tends to live in inland water with high mineral content [37]. P. parvum, sometimes known as "golden alga", has been reported to cause huge fish kills.…”