Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs. dried blood spot sample collection for malaria molecular surveillance.
Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs. dried blood spot sample collection for malaria molecular surveillance.
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DOI：
10.1186/s12936-022-04122-9
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发表时间：
2022-03-15
期刊：
影响因子：
3
通讯作者：
Koepfli C
中科院分区：
文献类型：
--
作者：
Holzschuh A;Koepfli C
分子和基因组监测正越来越多地用于跟踪疟疾控制和消除工作。血液样本可以作为全血收集，并在-20 °C下储存，直到DNA提取，或者作为干血斑（DBS）收集，从而避免了冷链的需要。尽管这两种方法都被广泛使用，但缺乏对血液样品保存方法如何影响分子诊断的检测限（LOD）和回收用于下游应用的DNA比例的系统比较。使用恶性疟原虫培养物样品的稀释系列，比较了基于旋转柱、磁珠、吐温-Chelex和直接PCR的提取方法（未预先提取）对全血和干血斑（DBS）的提取。通过qPCR和液滴数字PCR（ddPCR）定量提取的DNA。全血的DNA回收率比DBS高5- 10倍，导致两种提取方法的LOD比DBS低2- 3倍。对于全血，当从全血中提取时，基于磁珠的方法导致88-98%的DNA回收率，而基于旋转柱的方法为17-33%。对于DBS的提取，基于磁珠的方法导致8-20%的DNA回收率，而基于旋转柱的方法导致仅2%的DNA回收率。Tween-Chelex法的DNA回收率为15-21%，比其他方法的DNA回收率高上级，且灵敏度高于全血提取法。发现直接PCR方法对于全血和DBS两者具有最低的总体LOD。当比较基于分子方法的流行率估计值和选择其他分子监测应用的采样方案时，需要考虑LOD和DNA产量的显著差异。在线版本包含补充材料，可通过10.1186/s12936-022-04122-9获得。
Molecular and genomic surveillance is becoming increasingly used to track malaria control and elimination efforts. Blood samples can be collected as whole blood and stored at − 20 °C until DNA extraction, or as dried blood spots (DBS), circumventing the need for a cold chain. Despite the wide use of either method, systematic comparisons of how the method of blood sample preservation affects the limit of detection (LOD) of molecular diagnosis and the proportion of DNA recovered for downstream applications are lacking. Extractions based on spin columns, magnetic beads, Tween-Chelex, and direct PCR without prior extraction were compared for whole blood and dried blood spots (DBS) using dilution series of Plasmodium falciparum culture samples. Extracted DNA was quantified by qPCR and droplet digital PCR (ddPCR). DNA recovery was 5- to 10-fold higher for whole blood compared to DBS, resulting in a 2- to 3-fold lower LOD for both extraction methods compared to DBS. For whole blood, a magnetic bead-based method resulted in a DNA recovery rate of 88–98% when extracting from whole blood compared to 17–33% for a spin-column based method. For extractions from DBS, the magnetic bead-based method resulted in 8–20% DNA recovery, while the spin-column based method resulted in only 2% DNA recovery. The Tween-Chelex method was superior to other methods with 15–21% DNA recovery, and even more sensitive than extractions from whole blood samples. The direct PCR method was found to have the lowest LOD overall for both, whole blood and DBS. Pronounced differences in LOD and DNA yield need to be considered when comparing prevalence estimates based on molecular methods and when selecting sampling protocols for other molecular surveillance applications. The online version contains supplementary material available at 10.1186/s12936-022-04122-9.
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影响因子：
4.6
作者：
Nag S;Dalgaard MD;Kofoed PE;Ursing J;Crespo M;Andersen LO;Aarestrup FM;Lund O;Alifrangis M
通讯作者：
Alifrangis M
DOI：
10.1093/infdis/jiy223
发表时间：
2018-08-14
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
Aydemir O;Janko M;Hathaway NJ;Verity R;Mwandagalirwa MK;Tshefu AK;Tessema SK;Marsh PW;Tran A;Reimonn T;Ghani AC;Ghansah A;Juliano JJ;Greenhouse BR;Emch M;Meshnick SR;Bailey JA
通讯作者：
Bailey JA
影响因子：
4.6
作者：
Mahittikorn A;Masangkay FR;Kotepui KU;De Jesus Milanez G;Kotepui M
通讯作者：
Kotepui M
影响因子：
3
作者：
Baidjoe A;Stone W;Ploemen I;Shagari S;Grignard L;Osoti V;Makori E;Stevenson J;Kariuki S;Sutherland C;Sauerwein R;Cox J;Drakeley C;Bousema T
通讯作者：
Bousema T
影响因子：
3
作者：
Grossenbacher, Benjamin;Holzschuh, Aurel;Felger, Ingrid
通讯作者：
Felger, Ingrid