Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots.
Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots.
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DOI：
10.1186/1475-2875-11-339
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发表时间：
2012-10-08
期刊：
影响因子：
3
通讯作者：
Imwong M
中科院分区：
文献类型：
--
作者：
Hwang J;Jaroensuk J;Leimanis ML;Russell B;McGready R;Day N;Snounou G;Nosten F;Imwong M
在流行病学和临床调查中收集血液样本，然后作为在滤纸上晒干的血斑，通常在室温下储存，这已成为疟疾寄生虫进一步分子分析最受欢迎的材料来源之一。干燥的血斑通常被存档，以便用于寄生虫流行的进一步回顾性调查，或者作为新的遗传标记出现。然而，从储存了很长时间用于DNA扩增的干燥血斑中获得的模板的适用性尚不清楚。采用Chelex柱和Qiagen柱两种方法中的一种纯化方法，从显微镜下确诊的恶性疟原虫感染者12年间收集的267个存档血斑中提取DNA。这些模板针对三个长度和/或拷贝数不同的寄生虫位点进行了高度敏感的巢式PCR扩增。当针对疟原虫小亚基核糖体RNA的1.6 kb片段（初级扩增）时，在保存6年以上的样本中，恶性疟原虫的检测效率下降，对于保存10年以上的样本，检测效率达到非常低的水平。用qiagen提取的模板通常获得更多的阳性扩增。40个阴性qiagen提取模板中，有32个检测到恶性疟原虫，目标微卫星长度约为180bp。其余8个样本在高拷贝数（20 ~ 200）线粒体基因组238 bp的小区域内扩增呈阳性。从干燥的血斑中恢复的DNA片段的平均长度随着储存时间的延长而缩短。通过使用商业DNA纯化柱，DNA的回收率有所提高，特别是在较旧的样本中，但是在最初的血液采集后10年不太可能存在大于1.5 kb的目标，此时存在的DNA片段的平均长度可能在几百bp左右。总之，存档的干血点用于分子分析的效用随着储存时间的延长而降低。
Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known. DNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number. When a 1.6 kb fragment of the parasites’ small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted. The average length of DNA fragments that can be recovered from dried blood spots decreases with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time.
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影响因子：
64.8
作者：
Carlton, Jane M.;Adams, John H.;Silva, Joana C.;Bidwell, Shelby L.;Lorenzi, Hernan;Caler, Elisabet;Crabtree, Jonathan;Angiuoli, Samuel V.;Merino, Emilio F.;Amedeo, Paolo;Cheng, Qin;Coulson, Richard M. R.;Crabb, Brendan S.;del Portillo, Hernando A.;Essien, Kobby;Feldblyum, Tamara V.;Fernandez-Becerra, Carmen;Gilson, Paul R.;Gueye, Amy H.;Guo, Xiang;Kang'a, Simon;Kooij, Taco W. A.;Korsinczky, Michael;Meyer, Esmeralda V. -S.;Nene, Vish;Paulsen, Ian;White, Owen;Ralph, Stuart A.;Ren, Qinghu;Sargeant, Tobias J.;Salzberg, Steven L.;Stoeckert, Christian J.;Sullivan, Steven A.;Yamamoto, Marcio M.;Hoffman, Stephen L.;Wortman, Jennifer R.;Gardner, Malcolm J.;Galinski, Mary R.;Barnwell, John W.;Fraser-Liggett, Claire M.
通讯作者：
Fraser-Liggett, Claire M.
DOI：
10.1016/s0035-9203(99)90177-3
发表时间：
1999-01-01
影响因子：
2.2
作者：
Färnert, A;Arez, AP;do Rosário, V
通讯作者：
do Rosário, V
影响因子：
9.4
作者：
Polley, Spencer D.;Mori, Yasuyoshi;Sutherland, Colin J.
通讯作者：
Sutherland, Colin J.
DOI：
10.4269/ajtmh.2003.69.42
发表时间：
2003-07-01
影响因子：
3.3
作者：
Chaorattanakawee, S;Natalang, O;Patarapotikul, J
通讯作者：
Patarapotikul, J
影响因子：
30.8
作者：
Neafsey, Daniel E.;Galinsky, Kevin;Jiang, Rays H. Y.;Young, Lauren;Sykes, Sean M.;Saif, Sakina;Gujja, Sharvari;Goldberg, Jonathan M.;Young, Sarah;Zeng, Qiandong;Chapman, Sinead B.;Dash, Aditya P.;Anvikar, Anupkumar R.;Sutton, Patrick L.;Birren, Bruce W.;Escalante, Ananias A.;Barnwell, John W.;Carlton, Jane M.
通讯作者：
Carlton, Jane M.