Zheng Xiang , Danlin Li , Siqi Wang , Ting Shen , Wen He , Mier Li , Weilin Zeng , Xi Chen , Yanrui Wu , Liwang Cui , Zhaoqing Yang
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引用次数: 2
摘要
一种快速、简单、简单、高效、廉价的方法从滤纸上收集的疟原虫中提取DNA,对分子监测非常有用。DNA的质量和数量对分子诊断和分析至关重要。在这里,我们开发了一种简单的碱裂解法,用于在滤纸上从血液样本中提取DNA。结果表明,10–50 mM NaOH和去离子水都能在较高的寄生虫血症下有效分离寄生虫DNA,如成功的PCR扩增所示,而在0.01%的寄生虫血症时,10 mM NaOH裂解条件产生了最好的结果。此外,通过该方法提取的DNA被成功地用于扩增>;2000 bp。该方法成功地从1µl血液中提取了DNA,寄生虫血症低至0.0001%(相当于5个寄生虫/µl)。通过10mM NaOH裂解法分离的DNA在4°C或−20°C下储存12个月后稳定产生PCR产物。这些结果表明,这种碱裂解方法简单、有效、灵敏、廉价,可以从滤纸上干燥的血点中分离稳定的疟原虫DNA。
A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples
A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.
期刊介绍:
The journal provides a medium for rapid publication of investigations of the molecular biology and biochemistry of parasitic protozoa and helminths and their interactions with both the definitive and intermediate host. The main subject areas covered are:
• the structure, biosynthesis, degradation, properties and function of DNA, RNA, proteins, lipids, carbohydrates and small molecular-weight substances
• intermediary metabolism and bioenergetics
• drug target characterization and the mode of action of antiparasitic drugs
• molecular and biochemical aspects of membrane structure and function
• host-parasite relationships that focus on the parasite, particularly as related to specific parasite molecules.
• analysis of genes and genome structure, function and expression
• analysis of variation in parasite populations relevant to genetic exchange, pathogenesis, drug and vaccine target characterization, and drug resistance.
• parasite protein trafficking, organelle biogenesis, and cellular structure especially with reference to the roles of specific molecules
• parasite programmed cell death, development, and cell division at the molecular level.