Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool
Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool
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DOI：
10.1371/journal.pntd.0007778
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发表时间：
2019-10-01
影响因子：
3.8
通讯作者：
Levecke, Bruno
中科院分区：
文献类型：
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作者：
Ayana, Mio;Cools, Piet;Levecke, Bruno
DNA的提取和保存是任何核酸扩增试验（NAAT）成功的关键，但它仍然对土壤传播蠕虫（STHs）提出了挑战，包括蚓类蛔虫、毛线虫和两种钩虫（美洲钩虫和十二指肠钩虫）。在本研究中，我们评估了不同的DNA提取和保存方案对粪便中sth特异性DNA扩增的影响。方法和主要发现在第一个实验中，使用两种商业试剂盒从37份粪便样本中提取DNA，这些粪便样本含有不同数量的毛毛滴虫和美洲滴虫，分别有和没有事先的头部敲打步骤。采用qPCR方法对毛滴虫和美洲滴虫的DNA浓度进行了测定。结果显示，两种DNA提取试剂盒的DNA浓度存在明显差异，这在两种STHs中都有所不同。他们还指出，增加头部跳动的步骤大大提高了DNA的恢复，特别是当fec很高的时候。在第二个实验中，将20份不同卵数的粪便样本分别保存在96%乙醇、5%重铬酸钾或RNA中，并在4℃下保存65、245和425天。DNA的提取使用DNeasy血液和组织试剂盒与头部跳动步骤。在乙醇中保存的粪便样本证明，随着FEC的增加，产生更高的DNA浓度，尽管粪便样本在所有防腐剂中似乎都是稳定的。结论DNA提取试剂盒的选择对NAATs结果有显著影响。鉴于头部敲打的明显好处以及我们对乙醇（长期）保存的验证，我们建议任何粪便取样和DNA提取方案都应采用该方案的这些方面，以进行下游基于naat的STHs检测和定量。
BackgroundA DNA extraction and preservation protocol that yields sufficient and qualitative DNA is pivotal for the success of any nucleic acid amplification test (NAAT), but it still poses a challenge for soil-transmitted helminths (STHs), including Ascaris lumbricoides, Trichuris trichiura and the two hookworms (Necator americanus and Ancylostoma duodenale). In the present study, we assessed the impact of different DNA extraction and preservativation protocols on STH-specific DNA amplification from stool.Methodology and principal findingsIn a first experiment, DNA was extracted from 37 stool samples with variable egg counts for T. trichiura and N. americanus applying two commercial kits, both with and without a prior bead beating step. The DNA concentration of T. trichiura and N. americanus was estimated by means of qPCR. The results showed clear differences in DNA concentration across both DNA extraction kits, which varied across both STHs. They also indicated that adding a bead beating step substantially improved DNA recovery, particularly when the FECs were high. In a second experiment, 20 stool samples with variable egg counts for A. lumbricoides, T. trichiura and N. americanus were preserved in either 96% ethanol, 5% potassium dichromate or RNA later and were stored at 4 degrees C for 65, 245 and 425 days. DNA was extracted using the DNeasy Blood & Tissue kit with a bead beating step. Stool samples preserved in ethanol proved to yield higher DNA concentrations as FEC increased, although stool samples appeared to be stable over time in all preservatives.ConclusionsThe choice of DNA extraction kit significantly affects the outcome of NAATs. Given the clear benefit of bead beating and our validation of ethanol for (long-term) preservation, we recommend that these aspects of the protocol should be adopted by any stool sampling and DNA extraction protocol for downstream NAAT-based detection and quantification of STHs.