Data_Sheet_1_Development of a quadruplex PCR amplicon next generation sequencing assay for detection and differentiation of Bartonella spp..PDF

The genus Bartonella includes a group of species that are associated with a wide range of mammalian species, including human. It is challenging to detect all Bartonella species using a single molecular target due to its high genetic diversity. To solve this issue, we developed a quadruplex PCR amplicon sequencing assay using next-generation sequencing (NGS) technology for the detection and differentiation of Bartonella species. Our objective was to obtain the specific sequences of a minimum of two of the four target genes as confirmation of the identity of a particular Bartonella species using the assay. Four pairs of primers targeting specific regions on gltA, groEL, rpoB, and ssrA were evaluated for their capability of differentiating Bartonella species individually and collectively by performing singular PCR amplicon sequencing and quadruplex PCR amplicon sequencing. Using the quadruplex PCR amplicon sequencing, 24 Bartonella reference species were tested, all of which were successfully differentiated by at least two targets. Bartonella species were accurately identified from the artificially mixed DNA templates developed to simulate coinfections. The limit of detection was determined to be 1 fg based on testing a series of 10-fold dilutions of DNA from the Bartonella species. Testing of high DNA concentrations of 19 non-Bartonella species showed high specificity with none of the non-Bartonella species misclassified as Bartonella. Finally, the assay was evaluated by testing DNA extracts from field-collected body lice (Pediculus humanus humanus) and Norway rats (Rattus norvegicus): Bartonella quintana was detected and confirmed by three targets in the lice and Bartonella tribocorum was detected and confirmed by two targets in the rats. These results demonstrated that Bartonella species could be accurately and rapidly detected and differentiated into different tissue types using the quadruplex sequencing assay.

巴尔通体属（Bartonella）包含一类与包括人类在内的多种哺乳动物宿主相关的菌种。由于该属菌种具有高度的遗传多样性，仅依靠单一分子靶标难以实现所有巴尔通体菌种的全面检测。为解决这一难题，本研究基于下一代测序（next-generation sequencing, NGS）技术开发了一种四重PCR扩增子测序检测法，用于巴尔通体菌种的检测与区分。本研究的目标为：通过该检测方法获取4个靶标基因中至少2个的特异性序列，以此确认特定巴尔通体菌种的种属身份。我们针对gltA、groEL、rpoB及ssrA基因的特异性区域设计了四对引物，并通过单重PCR扩增子测序与四重PCR扩增子测序，分别评估了各引物对以及多引物组合区分巴尔通体菌种的能力。借助该四重PCR扩增子测序方法，我们对24株巴尔通体参考菌种进行了检测，所有菌种均可通过至少两个靶标实现有效区分。从模拟混合感染的人工构建DNA模板中，可准确鉴定出巴尔通体菌种。通过对巴尔通体菌种DNA进行一系列10倍梯度稀释检测，本方法的检测限确定为1飞克（fg）。对19株非巴尔通体菌种的高浓度DNA进行检测，结果显示该方法具有极高的特异性，无任何非巴尔通体菌种被误判为巴尔通体。本研究最终通过野外采集的人体虱（Pediculus humanus humanus）与褐家鼠（Rattus norvegicus）的DNA提取物对该检测方法进行了验证：在体虱样本中检测到五日热巴尔通体（Bartonella quintana），并通过3个靶标得到确认；在褐家鼠样本中检测到特里科伦巴尔通体（Bartonella tribocorum），并通过2个靶标得到确认。本研究结果证实，采用该四重测序检测法，可准确、快速地检测并区分巴尔通体菌种，且适用于不同组织类型样本。