Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction
Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction
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DOI：
10.1016/s0035-9203(99)90177-3
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发表时间：
1999-01-01
影响因子：
2.2
通讯作者：
do Rosário, V
中科院分区：
文献类型：
--
作者：
Färnert, A;Arez, AP;do Rosário, V
聚合酶链反应（PCR）现在被广泛用于疟疾研究中的现场样本分析。然而，很少有关于由于实地方法而失去灵敏度的报道。因此，进行了与血液采样（抗凝剂、培养基、滤纸）、储存（温度、时间和立即裂解）和处理（重复解冻和冷冻）相关的研究。PCR不受柠檬酸盐和EDTA的影响，但部分受到肝素的抑制（抑制作用在最佳浓度下被肝素酶逆转）。当储存在30 ℃和60%湿度下时，滤纸上收集的样品显示出显著低100倍的灵敏度（与收集后立即冷冻的对照样品相比）;并且纸张质量似乎很关键。未经处理的全血在4 ℃、20 ℃或30 ℃下储存很少会导致灵敏度的任何损失。与保持冷冻直到DNA提取的血液相比，重复解冻通常导致10倍的灵敏度损失。抗疟药的存在并不明显影响敏感性。我们的结论是，收集和储存的血液样本的模式可能会影响PCR检测疟疾寄生虫的灵敏度，这可能是至关重要的研究，包括个人与低寄生虫血症，混合感染和不同环境的数据比较。
Polymerase chain reaction (PCR) is now widely used in malaria research for analysis of field samples. However, little has been reported regarding loss of sensitivity due to field methodology. Therefore, studies were carried out in relation to blood sampling (anticoagulants, culture medium, filter paper), storage (temperature, time and immediate lysis) and handling (repeated thawing and freezing). The PCR was unaffected by citrate and EDTA but partly inhibited by heparin (inhibition was reversed by heparinase at optimal concentrations). Samples collected on filter paper showed a significant 100-fold lower sensitivity (compared to control samples frozen immediately after collection) when stored at 30 degrees C and 60% humidity; and the paper quality appeared to be critical. Storage of unprocessed whole blood at 4 degrees C, 20 degrees C or 30 degrees C rarely resulted in any loss of sensitivity. Repeated thawing generally resulted in 10-fold loss of sensitivity compared to blood kept frozen until DNA extraction. The presence of antimalarial drug did not apparently affect sensitivity. We conclude that the mode of collection and storage of blood samples may influence the sensitivity of detection of malaria parasites by PCR This may be critical in studies including individuals with low parasitaemia, mixed infections and comparison of data from different settings.