Table_2_Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children.xlsx

ObjectivesTo develop a rapid and low-cost method for 16S rDNA nanopore sequencing.
MethodsThis was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the experimental procedure. Twenty-one common pulmonary bacteria (7 reference strains, 14 clinical isolates) and 94 samples of bronchoalveolar lavage fluid from children with severe pneumonia were tested. Results indicating low-abundance pathogenic bacteria were verified with the polymerase chain reaction (PCR). Further, the results were compared with those of culture or PCR.
ResultsThe turnaround time was shortened to 6~8 hours and the reagent cost of DNA preparation was reduced by employing a single reaction adding barcodes to the 16S primer in advance. The accuracy rate for the 21 common pulmonary pathogens with an abundance ≥ 99% was 100%. Applying the culture or PCR results as the gold standard, 71 (75.5%) of the 94 patients were positive, including 25 positive cultures (26.6%) and 52 positive quantitative PCRs (55.3%). The median abundance in the positive culture and qPCR samples were 29.9% and 6.7%, respectively. With an abundance threshold increase of 1%, 5%, 10%, 15% and 20%, the test sensitivity decreased gradually to 98.6%, 84.9%, 72.6%, 67.1% and 64.4%, respectively, and the test specificity increased gradually to 33.3%, 71.4%, 81.0%, 90.5% and 100.0%, respectively.
ConclusionsThe nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children.

研究目标：开发一种用于16S核糖体DNA（16S rDNA）纳米孔测序（nanopore sequencing）的快速低成本方法。
研究方法：本研究为一项针对16S rDNA纳米孔测序方法的前瞻性研究。我们通过在16S引物中添加条码（barcode）以降低试剂成本并简化实验流程，开发了纳米孔条码标记16S测序法。本研究共纳入21株常见肺部细菌（7株参考菌株、14株临床分离株）以及94例儿童重症肺炎患者的支气管肺泡灌洗液样本进行检测。对于提示低丰度致病菌的测序结果，采用聚合酶链反应（polymerase chain reaction, PCR）进行验证，并将测序结果与培养法或PCR检测结果进行对比。
研究结果：通过预先在16S引物中添加条码的单反应体系，本方法将周转时间缩短至6~8小时，并降低了DNA制备的试剂成本。对于丰度≥99%的21株常见肺部病原菌，本方法的鉴定准确率达100%。以培养法或PCR检测结果作为金标准，94例患者中71例（75.5%）检测结果呈阳性，其中培养法阳性25例（26.6%），定量聚合酶链反应（quantitative PCR, qPCR）阳性52例（55.3%）。培养阳性样本与qPCR阳性样本的丰度中位数分别为29.9%和6.7%。当丰度阈值分别提高1%、5%、10%、15%和20%时，检测灵敏度逐渐降至98.6%、84.9%、72.6%、67.1%和64.4%，而检测特异性则逐渐升至33.3%、71.4%、81.0%、90.5%和100.0%。
研究结论：该纳米孔条码标记16S测序法可快速鉴定儿童细菌性肺炎的病原菌。