A. salmonicida is widespread distributed in nature and is an opportunistic pathogen that caused significant economic losses in aquaculture. To detect A. salmonicida infections, real-time PCR (RT-qPCR) and antibody-coated gold nanoparticles immunoassay methods have been developed since 2006 (Balcazar et al., 2007; Bartkova et al., 2017; Saleh et al., 2011). Although these researches are fairly sensitive and fast, they are mainly limited to laboratory settings owing to the expensive and complex facility required (Hou et al., 2017). As a result, a simple point-of-need diagnostic test is critical for limiting A. salmonicida spread and controlling outbreaks. In this context, RPA and RPA-LFD were established and validated for the rapid detection of A. salmonicida, both of which were reliable and highly sensitive (Faye et al.; Fu et al., 2019). What is more important, the specificity, detection restrict, and application feasibility of the established RPA and RPA-LFD assays have been well estimated.
To address the need for fast, and economical detection in the field, other isothermal researches like LAMP assay is also established for the detection of A. salmonicida (Zhou et al., 2021). However, RPA and RPA-LFD have several advantages compared to LAMP. Firstly, RPA and RPA-LFD are more easily designed, and require only one set of primers and one probe. Whereas LAMP applies a larger set of 4–6 primers to identify 6–8 different regions in the target sequence (Song et al., 2021). Secondly, RPA and RPA-LFD can be accomplished within 20 min, whereas LAMP takes 1-1.5 h (Srisrattakarn et al., 2020; Y. Yang et al., 2016). Finally, the RPA and RPA-LFD reaction can be carried out at invariant and relatively low temperatures ranging from 35 to 45°C and can be cultured with body temperature alone, thus easier to facilitate field application (Geng et al., 2019; Liu et al., 2019). By contrast, LAMP demands a higher temperature ranging from 62 to 67°C.
To ensure the specificity and sensitivity of amplification, we considered the design principles of the RPA primers and probes, containing the nucleotide composition (especially at the 5' end), the primer sequence, and the interaction between the forward primer, probe and the reverse primer (Chen et al., 2020; Hu et al., 2019). The length of the RPA primer is approximately 35 bp, which avert guanine duplicates at the 5' terminal. The primers were 30 bp and the GC content was approximately 45% in this research. Furthermore, although the present RPA kit can amplify DNA larger than 500bp, the optimal amplification size of RPA for higher sensitivity and speed is approximately 200bp. In our research, the amplification size of RPA and RPA-LFD were 191 bp and 112 bp which may be the account for the higher sensitivity. Applications of better RPA primers should be bound to high-quality probes, the internal cleavage site of the RPA probe is separated by a dT-fluorophore and a corresponding dT-quencher group, which is divide by exonuclease, and the accumulated fluorescence can be tested by a real-time fluorophore detector. Furthermore, the RPA-LFD probe was labeled with FAM antigenic at 5' end, and the 3' end polymerase extension blocking group was labeled with C3 spacer, which is different from the RPA probe (Y. Yang et al., 2016). Double-label amplification can be tested on an LFD device after amplification.
For molecular detection methods, specificity should be considered, so that the selection of detection targets is very important (Dong et al., 2019). The previous researches such as real-time PCR and LAMP have been selected the vapA gene as targets gene to detect A. salmonicida, the main function of the vapA gene is to encode a layer A protein and it is also named as the A-layer gene. Among them, it is reported that vapA gene almost contains the A. salmonicida and A. salmonicida subsp, the level of evolutionary variation of vapA gene sequence determines that it can be used to detect A. salmonicid, so we selected it as our target gene in this research. When using RPA and RPA-LFD to detect A. salmonicida, the specificity of primers and probes is very critical. In terms of methodology, the RPA and RPA-LFD detection methods produced in this researches have dual specificity, namely primer specificity and probe specificity. In the design of primers and probes, the related sequences displaying excellent similarity with the vapA from different A. salmonicida. The specific sequence regions of A. salmonicida were recognized and further applied to design specific primers and probes. Therefore, RPA and RPA-LFD with the primers and probes were specific, which ensures the specificity of RPA and RPA-LFD analysis (Fig. 5a and 5b). In addition, the sequence analysis showed that the vapA sequences of different A. salmonicida subsp deposited in GenBank exhibited a high similarity (Fu et al., 2019). Thus, the designed primers and probe could be applied in testing some A. salmonicida subsp such as A. salmonicida subsp. achromogenes and A. salmonicida subsp. Masoucida (Fig. 4a and 4b).
In terms of analytical sensitivity, the sensitivity of the RPA and RPA-LFD detection as reported here was sufficient (Dai et al., 2019). In the sensitivity evaluation using plasmid, the real-time PCR assay has been established to display higher sensitivities detecting as low as 5.6×102 copies/uL (Lian et al., 2020). However, the detection lower limit for this RPA and RPA-LFD method in this researches were 102 and 1 copies/uL (Fig. 6a and 6b), which were at least 5.6 and 560 times higher than that of the qPCR method. Notably, the high sensitivity and specificity of RPA-LFD contribute to the full utilization of gold-labeled anti-FAM antibodies and anti-biotin antibodies to simultaneously capture RPA products (Hou et al., 2017). Theoretically, RPA can test a single copy of a DNA target, so there is room for further optimization and development, especially to pay close attention to different target regions, probe and primer combinations and concentrations to improve detection sensitivity (Rostron et al., 2019).
To imitate the field detecting condition, we also assessed the sensitivity of the RPA and RPA-LFD by using DNA collected from tissues (liver, spleen, intestine) spiked with the A. salmonicida (Wu et al., 2019). The limit of detection (LOD) of the RPA and RPA-LFD turned out to be 1.2×101 CFU/mL and 1.2 CFU/mL per reaction of A. salmonicida (Table 4). The above results indicated that RPA-FLD assay was more sensitive than RPA assay from spiked tissues (liver, spleen, intestine). To the best of our knowledge, the difference in sensitivity between the two methods may be caused by the combination of different primers and probes. The nfo reaction mechanism produces double-labeled reporter at the same time as amplification, which requires minimal amplification post-processing (Hu et al., 2019). PCR and Real-time PCR testing of A. salmonicida is currently regarded as the definitive assay for human diagnostics. However, the LOD for PCR and real-time PCR in fish tissues was 250 CFU/mL and 32 CFU/mL (Balcazar et al., 2007; Onuk et al., 2010), respectively. Their sensitivity was much lower than the RPA and RPA-LFD assays. As a result, the RPA and RPA-LFD assays established here have potential applications.