Objective: To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods.
Methods: The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers' urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit).
Results: An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers' urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01).
Conclusions: A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers' urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.
[摘要] 目的建立尿液中外源添加的日本血吸虫小分子DNA片段提取方法, 评价不同方法处理尿液后的提取效果。 方法以日本血吸虫SjG28基因片段作为靶序列, 通过PCR扩增靶序列上的81 bp小分子DNA片段, 经测序鉴定后作为 拟添加到尿液样本中的外源小分子DNA片段。以SjG28为靶基因设计引物及探针, 建立实时荧光定量PCR(qPCR)方 法;将外源小分子DNA片段以10为梯度倍比稀释后进行扩增, 评价该方法的敏感性; 以日本血吸虫、曼氏血吸虫、埃及血 吸虫、巴贝虫、十二指肠钩口线虫、华支睾吸虫、卫氏并殖吸虫基因组DNA为模板进行检测, 评价该方法的特异性。将外 源小分子DNA片段添加至人工尿液及健康人尿液后, 经调整pH值、离心、浓缩等处理后, 采用QIAmp Viral RNA Mini Kit (Qiagen试剂盒) 、BIOG游离DNA提取试剂盒(BIOG试剂盒)提取尿液中的外源小分子DNA片段, 比较不同处理方式及 提取方法的效果。 结果成功制备81 bp外源日本血吸虫小分子DNA片段, 建立的qPCR法最低能检测到100拷贝小L 的81 bp外源日本血吸虫小分子DNA片段。以上述7种寄生虫DNA为模板进行检测, 仅日本血吸虫基因组DNA模板有 荧光信号值增长。调整人工尿液pH值为5、6、7、8, 采用Qiagen试剂盒提取其中的外源日本血吸虫小分子DNA片段回收 率分别(49.12 ±2.09)% (84.52 ±4.96)% (89.38 ±3.32)%和 (87.82 ±3.90)%;采用 BIOG 试剂盒提取小分子 DNA 片段回 收率分别为(2.30±0.07)%、 (8.11±0.26)%、 (13.35±0.61)%、 (20.82±0.68)%, 两种方法提取回收率差异均有统计学意 义(t= 38.702、26.955、39.042、29.571, P均<0.01)。采用Qiagen试剂盒提取人工尿液, pH值为5时核酸回收率最低(P均< 0.05); pH值为6、7、8时, 核酸回收率差异均无统计学意义(P均>0.05)。人工尿液[(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t= 12.033, P<0.05)]、健康人尿液[(31 165 ± 1 017)拷贝/μL vs.(28 471 ± 818)拷贝/μL; t = 23.164, P<0.05]经离心后, 外 源小分子DNA片段回收效果均降低;使用10K离心浓缩管浓缩人工尿液、使用100K离心浓缩管浓缩健康人尿液均能有 效提高Qiagen试剂盒回收效果(P均< 0.01)。 结论成功建立了尿液中外源日本血吸虫小分子DNA片段的提取方法, Qiagen试剂盒提取效果较好。将尿液样本pH值调整至6 ~ 8、使用100K离心浓缩管浓缩健康人尿液样本均可提高提取 效果。.
Keywords:
Cell-free DNA Short DNA fragment; Real-time fluorescent quantitative PCR assay;