Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.
Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.
复制标题
DOI：
10.1186/1475-2875-12-272
复制
发表时间：
2013-08-02
期刊：
影响因子：
3
通讯作者：
Bousema T
中科院分区：
文献类型：
--
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
通报和评估疟疾控制工作依赖于对当地传播动态的了解。血清学和分子工具已表现出在低流行环境下量化传播强度的高度敏感性，而传统方法的敏感性有限。滤纸血斑通常用作 DNA 和抗体的来源。为了提高疟疾调查的操作实用性，提出了一种结合 DNA 提取和抗体洗脱的方法。作为肯尼亚高地大型横断面调查的一部分，收集了滤纸血点。使用皂苷/螯合法提取DNA。 DNA 提取过程中第一次洗涤的洗脱液用于抗体检测，并与之前验证的抗体洗脱程序进行比较。通过疟疾抗原顶膜抗原 1 (AMA-1) 和裂殖子表面蛋白 1 (MSP-142) 的总 IgG ELISA 评估抗体洗脱效率。确定了巢式 18S rRNA 和细胞色素 b PCR 测定的灵敏度以及双层滤纸材料对 PCR 灵敏度的影响。使用发光和荧光报告分析来检查细胞材料和抗体在整个滤纸血点中的分布。结合抗体/DNA 提取技术后测量的抗体水平与标准抗体洗脱后测量的抗体水平密切相关 (p<<0.0001)。 AMA-1 和 MSP-142 的抗体水平普遍略低（11.3-21.4%），但年龄-血清流行率模式无法区分。在不同的 PCR 检测中，寄生虫阳性样本的比例范围为 12.9% 至 19.2%。尽管不同 PCR 检测的结果非常一致，但没有一种检测检测到所有寄生虫阳性个体。对于所有检测，用于 DNA 提取的双重滤纸材料提高了灵敏度。细胞和抗体材料的浓度在整个血点中分布不均匀。 DNA 提取和抗体洗脱相结合是一种具有操作吸引力的方法，可用于高通量评估累积的疟疾暴露和流行环境中当前的感染流行率。抗体流行率的估计不受提取和洗脱组合程序的影响。目的基因的选择以及用于 DNA 提取的滤纸材料的数量和来源对 PCR 灵敏度有显着影响。
Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots. Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.
登录
查看更多内容
一种针对线粒体基因组的新型单扩增 PCR 在诊断挪威卑尔根回国旅行者的疟疾方面具有高度敏感性和特异性。
影响因子：
3
作者：
Haanshuus CG;Mohn SC;Mørch K;Langeland N;Blomberg B;Hanevik K
通讯作者：
Hanevik K
影响因子：
6.4
作者：
Bousema, Teun;Drakeley, Chris;Gosling, Roly
通讯作者：
Gosling, Roly
影响因子：
3.7
作者：
Cook, Jackie;Kleinschmidt, Immo;Drakeley, Chris J.
通讯作者：
Drakeley, Chris J.
热点干预措施对疟疾传播的影响：整群随机对照试验的研究方案。
影响因子：
2.5
作者：
Bousema T;Stevenson J;Baidjoe A;Stresman G;Griffin JT;Kleinschmidt I;Remarque EJ;Vulule J;Bayoh N;Laserson K;Desai M;Sauerwein R;Drakeley C;Cox J
通讯作者：
Cox J
影响因子：
3
作者：
Hwang J;Jaroensuk J;Leimanis ML;Russell B;McGready R;Day N;Snounou G;Nosten F;Imwong M
通讯作者：
Imwong M