Optimizing rpoB metabarcoding for low-concentration bacterial DNA in eukaryotic matrices using nested PCR: application to insect microbiota. Nested PCR strategy for rpoB metabarcoding studies

In recent years, microbial ecosystems have been mainly studied using 16S rRNA gene metabarcoding. However, this strategy does not provide sufficient taxonomic resolution and often fails to determine the bacterial species. The aim of this work was to develop a novel method to improve the amplification of samples with low bacterial DNA concentrations embedded in eukaryotic matrices, while allowing to keep species-level taxonomic resolution. To this end, we optimized a metabarcoding procedure by combining nested PCR and targeting of a region of the rpoB housekeeping gene. First, we validated our method using in silico approaches. Then, we tested it on commercial and custom-made mock samples. Finally, we applied this procedure to whole larvae of the lepidopteran insect Spodoptera frugiperda collected in the field in Senegal , and to their oral secretions (OS). We demonstrated that rpoB nested PCR outperforms rpoB direct PCR for the detection of bacterial amplicon sequence variants ASVs in low-concentration bacterial DNA samples (OS samples) or bacterial DNA embedded in complex insect matrices (larvae samples), while allowing species-level resolution, unlike the short amplicon from 16S rRNA genes, which is limited to genus-level identification. Finally, we propose a new strategy to facilitate the description of insect-associated microbiota, which could be extended to other host-associated microbiota.

近年来，微生物生态系统的研究多采用16S rRNA基因宏条形码测序（16S rRNA gene metabarcoding）技术。但该技术的分类学分辨率有限，往往无法精准鉴定细菌物种。本研究旨在开发一种全新方法，在保留物种水平分类分辨率的前提下，优化嵌于真核生物基质中的低浓度细菌DNA样本的扩增流程。为此，我们结合嵌套式PCR（nested PCR）与rpoB持家基因（rpoB housekeeping gene）的区域靶向扩增，优化了一套宏条形码测序流程。首先，我们通过计算机模拟（in silico）方法验证了该方法的有效性；随后，利用商业化及定制化的模拟样本对其进行了性能测试；最后，将该流程应用于在塞内加尔野外采集的鳞翅目昆虫草地贪夜蛾（Spodoptera frugiperda）的完整幼虫及其口腔分泌物（oral secretions, OS）。研究证实，针对低浓度细菌DNA样本（即口腔分泌物样本）或嵌于复杂昆虫基质中的细菌DNA样本（即幼虫样本）的细菌扩增子序列变体（amplicon sequence variants, ASVs）检测中，rpoB嵌套PCR的检测性能优于rpoB直接PCR，且可实现物种水平的分类分辨率；而16S rRNA基因的短扩增子仅能实现属水平鉴定，无法达到物种级分辨率。最后，我们提出了一套全新的研究策略，可助力昆虫共生微生物组的解析工作，且该策略有望推广至其他宿主相关微生物组的研究中。