比较两种磁珠法核酸提取试剂在病原靶向二代测序（ptNGS）检测肺泡灌洗液（BF）和外周血（PB）样本的实验参数及检测结果差异，选择较优的核酸提取试剂并评估其在ptNGS检测中的适用性，为不同样本类型选择适用的核酸提取试剂提供参考。

选择两种代表性磁珠法核酸提取试剂盒KitA和KitB，对已知培养或实时荧光定量PCR（qPCR）结果的12例BF和8例PB样本分别取相同体积进行ptNGS检测，流程主要包括核酸提取、文库构建、上机测序和结果分析。采用Wilcoxon配对秩检验系统比较KitA与KitB在样本（BF和PB）检测过程中的实验参数（包括核酸提取总质量、吸光度（A）_260/280、文库浓度、样本测序数据质量指标Q30等）和病原检出拷贝数，以明确更适用于BF与PB样本ptNGS检测的磁珠法提取试剂。通过ptNGS检测的准确度、精密度、检出限和常规抗干扰能力进一步评估提取试剂盒的适用性。

KitA在BF的核酸提取总量显著高于KitB（(W=－66、P=0.001），而在PB的核酸提取总量与KitB差异无统计学意义（W=19、P=0.063）。KitB在BF和PB样本核酸的吸光度A_260/280显著优于KitA（BF：W=54、P=0.014，PB：W=21、P=0.031）。KitA与KitB提取核酸构建的文库浓度和片段大小差异均无统计学意义；KitA与KitB测序数据在Q30、引物二聚体百分比和Real align百分比等差异均无统计学意义（P均> 0.05）。KitA与KitB病原体检出数量和种类一致；KitA提取的BF检出病原体拷贝数显著高于KitB（W=－301、P < 0.001），但两者在PB中病原拷贝数差异无统计学意义（W=－3、P=0.844）。进一步选择更适用的核酸提取试剂盒KitA评估其在ptNGS检测BF和PB中的适用性，使用KitA进行ptNGS检测的阳性符合率为97.14%，漏检率为2.86%，总体符合率为95%；批内精密度和批间精密度均< 10%；细菌和真菌检出限均为100拷贝/ml，病毒检出限均为1 000拷贝/ml；样本中的非靶标核酸（包括人源核酸）对ptNGS定性结果无干扰。

KitA提取方式在BF的ptNGS检测中整体优于KitB，KitA和KitB在PB的ptNGS检测中整体实验参数如核酸提取总质量、文库浓度和测序数据质量指标Q30等基本一致，KitA在ptNGS检测中准确度、精密度、检出限和常规抗干扰能力的性能评估中均满足临床检测需求。

To evaluate and compare the experimental parameters and resultant differences of two magnetic bead-based nucleic acid extraction reagents in pathogen-targeted next-generation sequencing (ptNGS) for the detection of bronchoalveolar lavage fluid (BF) and peripheral blood (PB) samples, and to identify the more effective nucleic acid extraction reagent and assess its applicability in ptNGS detection, in order to offer guidance for the selection of appropriate nucleic acid extraction reagents tailored to various sample types.

Two representative magnetic bead-based nucleic acid extraction kits, designated as KitA and KitB were employed to conduct ptNGS detection on 12 BF samples and 8 PB samples, all of which had established culture or real-time fluorescence quantitative PCR (qPCR) results. An equal volume was utilized for each sample. The methodology encompassed several key stages, including nucleic acid extraction, library construction, sequencing and subsequent result analysis. The experimental parameters, including total nucleic acid extraction mass, absorbance (A) _260/280, library concentration and sample sequencing data quality index Q30; and pathogen detection copy numbers between KitA and KitB during the detection process of samples (BF and PB) were systematically compared by Wilcoxon signed-rank tests, and the magnetic bead extraction reagent which was more suitable for ptNGS detection of BF and PB samples were clarified. The suitability of the extraction kit was further evaluated by the accuracy, precision, detection limit and conventional anti-interference ability of ptNGS detection.

The total amount of nucleic acid extracted in BF by KitA was significantly higher than that of KitB (W=-66, P=0.001), but without significant difference in PB (W=19, P=0.063). However, the absorbance A_260/280 of KitB nucleic acid in BF and PB samples were significantly better than those of KitA (BF: W=54, P=0.014; PB: W=21, P=0.031). But the library concentration and fragment size of the libraries constructed by KitA and KitB nucleic acid were not significantly different; sequencing data quality metrics including Q30 scores, primer dimer percentage and percentage of Real aligne were not significantly different between the two extraction methods (all P > 0.05). The number and species of KitA and KitB detected in the pathogen were consistent. The pathogen copy number detected by KitA in BF was significantly higher than that of KitB (W=-301, P < 0.001), however, there was no statistically significant difference in pathogen copy number in PB between the two extraction methods (W=-3, P=0.844). Further, a more suitable nucleic acid extraction kit KitA was selected to evaluate its suitability for ptNGS detection in BF and PB. The positive concordance rate of ptNGS detected by KitA was 97.14%, the missed detection rate was 2.86%, and the overall concordance rate was 95%. Both intra-batch and inter-batch precision exhibited coefficients of variation below 10%. The limit of detection (LOD) was established at 100 copies/ml for bacterial and fungal targets, and 1 000 copies/ml for viral targets. Notably, non-target nucleic acids (including human-derived DNA/RNA) in the samples did not interfere with the qualitative results of ptNGS analysis.

For ptNGS detection in BF samples, Kit A demonstrated overall superior performance compared with Kit B across all evaluated parameters. In PB samples, both kits were consistent in overall experimental parameters, including total nucleic acid yield, library concentration and sequencing data quality indicators Q30. KitA meets the requirements of clinical testing in terms of accuracy, precision, detection limit and routine anti-interference capability in the performance evaluation of ptNGS detection