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While high efficiency and cost‐effectiveness are two merits of environmental DNA (eDNA) techniques for detecting aquatic organisms, the difficulty of designing species‐specific primers can result in significant expenditure of time and money. During the in silico stage of primer development, primer specificity is predicted with alignment techniques such as BLAST that is based on the number and position of the primer/nontarget template mismatches. However, we speculate that nonspecific amplification is influenced by additional parameters, which lead to inaccuracies of in silico prediction. We performed in vitro specificity tests for 38 species‐specific primers selected for seven fishes and six turtles, using single‐plex conventional PCR (cPCR). A subset of 12 primer pairs were further tested with SYBR Green‐based or TaqMan‐based single‐plex quantitative PCR (qPCR). We disentangle the relative importance of mismatch properties (types and positions), primer properties (length, GC content, and 3′ end stability), PCR conditions (template concentrations and annealing temperatures), and PCR technique (cPCR, TaqMan‐based, or SYBR Green‐based qPCR) in determining the occurrence of amplifications. We then compared the PCR outcomes with the specificity check under two stringency scenarios based on alignment (i.e., BLAST search). We conducted a total of 679 cPCR and 226 qPCR analyses, with 90% of the reactions tested with nontarget templates. Primer pairs predicted by Primer‐BLAST to be specific rarely showed such specificity during the in vitro testing. BLAST searches correctly predicted the outcomes of around 67% of cPCR and qPCR, but had low sensitivity in detection of nontarget amplification (29–57%). Primer specificity increased significantly with total number of mismatches and annealing temperature, but decreased with higher GC content in the primer sequence. Mismatches that consisted of A‐A, G‐A, and C‐C pairings exerted 56% stronger reduction in nonspecific amplification effects than other mismatches. To conclude, we show that the prediction of primer specificity based only on the number and position of mismatches can be misleading. Our findings can be applied to increase the efficiency of the in silico primer selection process to maintain the relatively high efficiency and cost‐effectiveness of eDNA techniques.
Bibliographical noteWe thank the two anonymous reviewers for their constructive comments and the editor for the review of the manuscript. We thank Amy Leung and Anthony Lau for providing suggestions, comments, and technical support. We are also grateful to Billy Hau, for spurring initiation of the work. This work was funded by the Hongkong and Shanghai Banking Corporation(and endorsed by the Agriculture, Fisheries and Conservation Department of Hong Kong SAR Government) under the Living Water & Community Revitalization—An Agricultural-led Action, Engagement and Incubation Programme at Lai Chi Wo and the Research Grants Council of the Hong Kong SpecialAdministrative Region, China (LU 23100216). Permits for possession of live turtles and their tissues for molecular work were granted by the Agricultural Fisheries and Conservation Department, Hong KongGovernment (AF GR CON 09/51 Pt.6). Ying Kin KenSo, Jonathan J. Fong, and David Dudgeon jointly wrote the manuscript. Ivan Pui Yin Lam and Ying Kin Ken So performed experiments. Ying Kin Ken So designed this study under David Dudgeon's supervision and performed data analyses.
- primer design
- primer specificity
- primer–template mismatch
- species-specific primer
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