Recombinase-aided amplification combined with lateral flow dipstick for the rapid detection of Amphidinium carterae

Abstract

In the context of global climate change, the frequency and duration of harmful algal blooms (HABs) due to eutrophic coastal waters have increased. HABs can cause marine ecological imbalance, massive socio-economic damage, and potential public health problems. Therefore, accurate and specific techniques that can rapidly detect harmful algae are critical to expedite early monitoring and prediction of HABs. Although some methods based on amplification have previously been described, limitations associated with limited popularity and high costs still exist. In this study, the partial sequence of nuclear ribosomal DNA from Amphinidium carterae was selected as the target region. The biotin-labeled recombinase-aided amplification (RAA) products and the 6-carboxyfluorescein-labeled probe were specifically conjugated, which formed double-labeled RAA products analyzed by lateral flow dipstick (LFD). A rapid RAA-LFD assay aimed at visually detecting A. carterae was established and optimized. The specificity and sensitivity of RAA-LFD were determined. The nucleic acid test with other control algal species yielded negative results, which indicated that RAA-LFD assay can specifically detect A. carterae. The detection limit of RAA-LFD assay for A. carterae was 8.49 ng μL −1 genomic DNA and 1 cell mL −1 . The whole detection process of RAA-LFD could be completed within 50 min. In conclusion, the proposed RAA-LFD assay may be promising for the efficient, specific, and rapid detection of A. carterae in monitoring efforts.

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

“…However, cryptic species cannot always be resolved with these genes, and more variable regions, such as the ITS regions, may be more appropriate. The ITS sequence was chosen as the target sequence of qPCR, RAA-LFD, LAMP-LFD, and RPA-LFD, as it is an effective molecular marker that is capable of detecting various HAB species accurately [26,27,49]. The choice of appropriate target sequences is essential for a molecular detection approach.…”

“…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.…”

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses

“…RAA-LFT has the same technical advantages as RPA-LFT. Another very attractive advantage of RAA-LFT is that its cost is much lower than that of RPA-LFT, which is ideal for the batch testing of samples [44].…”

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

“…Of the ten paper-based RAA articles incorporated in this review, all reported paper-based detection but did not show paper-based extraction or amplification. Nine articles reported colorimetric detection using tagged probes/primers and AuNPs [ 58 , 59 , 60 , 61 , 62 , 63 , 64 ]. For example, Xu et al used LFIA to detect Amphidinium carterae by implementing a species-specific primer tagged with biotin and a probe labeled with FAM [ 62 ].…”

“…Nine articles reported colorimetric detection using tagged probes/primers and AuNPs [ 58 , 59 , 60 , 61 , 62 , 63 , 64 ]. For example, Xu et al used LFIA to detect Amphidinium carterae by implementing a species-specific primer tagged with biotin and a probe labeled with FAM [ 62 ]. In a few cases, Cas12a reporter probes and AuNPs were implemented for detection [ 65 , 66 ].…”