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Pitfalls during in silico prediction of primer specificity for eDNA surveillance

机译：在伊纳监测的底漆特异性中陷阱中的陷阱

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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.
机译：虽然高效率和成本效益是用于检测水生生物的环境DNA（EDNA）技术的两个优点，但设计物种特异性引物的难度可能导致时间和金钱的显着支出。在底漆开发的硅阶段期间，预先预测引物特异性，例如诸如爆炸的对准技术，其基于底漆/非明显模板不匹配的数量和位置。然而，我们推测非特异性扩增受其他参数的影响，这导致硅预测的不准确性。使用单plex常规PCR（CPCR），我们对38种特异性引物进行了体外特异性试验。进一步用SybR绿色或基于TaqMan的单吡克隆定量PCR（QPCR）进一步测试12个引物对的子集。我们解开了不匹配性质（类型和位置），引物性质（长度，GC含量和3'稳定性），PCR条件（模板浓度和退火温度）和PCR技术（CPCR，Taqman，或基于CPCR的或）的相对重要性SYBR绿色基QPCR）在确定扩增发生时。然后，我们将PCR结果与基于对齐（即，BLAST搜索）的两个严格性情景下的特异性检查。我们共进行了679个CPCR和226 QPCR分析，其中90％的反应与Nontarget模板测试过。通过引物爆炸预测的引物对待特异性地在体外测试期间表现出这种特异性。 BLAST搜索正确预测了大约67％的CPCR和QPCR的结果，但在检测到Nontarget扩增的敏感性较低（29-57％）。引物特异性随着不匹配和退火温度的总数而显着增加，但在引物序列中具有较高的GC含量下降。由A-A，G-A和C-C配对组成的不匹配在非特异性扩增效果中施加56％，而不是其他不匹配。为了得出结论，只有基于错配的数量和位置的引物特异性的预测可能是误导性的。我们的研究结果可用于提高硅底漆选择过程中的效率，以保持EDNA技术的效率较高和成本效益。

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