Plasmodium falciparum: A simple, rapid method for detecting parasite clones in microtiter plates
Plasmodium falciparum: A simple, rapid method for detecting parasite clones in microtiter plates
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DOI：
10.1006/expr.1997.4156
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发表时间：
1997-06-01
影响因子：
2.1
通讯作者：
Taraschi, TF
中科院分区：
文献类型：
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作者：
Goodyer, ID;Taraschi, TF
恶性疟原虫的基因组定位和遗传操作产生的突变寄生虫的分析需要一种快速，灵敏的方法来识别选定的寄生虫克隆。本报告描述了先前描述的寄生虫乳酸脱氢酶（pLDH）试验（Makler等人，1993）用于此目的。克隆程序通常包括在96孔板中用未感染的红细胞稀释选定的寄生虫种群，每孔少于一个感染的红细胞。3周后，从每个孔制备Giemsa染色涂片并检查以确定哪些威尔斯孔含有寄生虫。使用该方法可检出低于0.1%的寄生虫血症。虽然这种方法是敏感的，但它非常耗时，严重限制了任何时候可以处理的板数。通过这种方法，单个96孔板可能需要至少一天的时间来分析。最近，已经描述了一种改进的寄生虫检测方法（Kirkman等人，1996年）。当寄生虫血症大于0.5%时，其利用RPMI 1640培养基的酚红指示剂的颜色从红色变为橙黄色。虽然比分析吉氏染色涂片快，但这种方法有一些缺点。首先，在苛刻的条件下培养寄生虫，因为培养基的黄色是低pH值的指示（由于乳酸的产生）。在低pH值条件下，寄生虫将处于葡萄糖剥夺状态。其次，该方法需要熟练的技术人员来检测细微的颜色变化。第三，该方法只能检测出超过0.5%的寄生虫血症（Kirkman等人，1996）。寄生虫特异性乳酸脱氢酶测定首先由Makler等人描述。（1993年）。pLDH试验对疟原虫酶具有特异性，不能检测红细胞LDH。其原理是疟疾LDH可以利用3-乙酰吡啶NAD（APAD）作为NAD类似物，而红细胞LDH不能（Makler和Hinrichs 1993）。测定试剂，氮蓝四唑（NBT）和硫酸苯乙酯（PES），从Sigma（St. Louis，MO）获得。Malstat试剂购自Flow，Inc.（波特兰，OR）。为了进行pLDH测定，将96孔板的每个孔中的20 μl感染的红细胞（1%血细胞比容）与100 μl Malstat试剂、10 μl 1 mg/ml NBT和10 μl 0.1mg/ml PES在20 ℃下混合30分钟。向各孔中加入100 μl 5%乙酸终止反应。在酶标仪上读取650 nm处的吸光度（A650）。已证明A650与Giemsa染色血涂片检测到的寄生虫血症成比例（Makler等人，1993）。为了测试pLDH测定用于鉴定疟疾克隆的效用，通过Wu等人的方法的修改转染恶性疟原虫（乙胺嘧啶敏感株3D 7）。（1995），用含有弓形虫二氢叶酸还原酶/胸苷酸合成酶基因的DNA构建体，所述基因侧翼为来自疟疾节相关富组氨酸蛋白（KAHRP）的调节区（Lanzer等，1992）。细胞在15 ng/ml乙胺嘧啶存在下生长5周，之后分离抗药性寄生虫。将这些寄生虫稀释并转移至96孔微量滴定板中，使用Modular Chamber Incubator（Vanguard，Neptune，NJ）将其保持在低氧气氛（1% O2、5% CO2、94% N2）中。1周后更换培养基。再过一周（接种后14天）后，对微量滴定板中的每个样品进行pLDH测定。部分威尔斯孔呈弱阳性（A650 ≤ 0.1）。换了培养基，细胞...
The genomic mapping of Plasmodium falciparum and the analysis of mutant parasites produced by genetic manipulation requires a rapid, sensitive method to identify selected parasite clones. This report describes the utilization of a previously described parasite lactate dehydrogenase (pLDH) assay (Makler et al. 1993) for this purpose. The cloning procedure generally consists of diluting the selected parasite populations with uninfected erythrocytes in 96-well plates at less than one infected erythrocyte per well. After a 3-week period, a Giemsa-stained smear is prepared from each well and examined to determine which wells contain parasites. Parasitemias lower than 0.1% are detectable using this method. While this method is sensitive, it is very time consuming, severely limiting the number of plates that can be processed at any time. A single 96-well plate can take at least a day to analyze by this method. Recently, an improved method for parasite detection has been described (Kirkman et al. 1996). It utilizes the change in color of the phenol red indicator of RPMI 1640 culture medium from red to orange–yellow when the parasitemia is greater than 0.5%. Although faster than analyzing Giemsastained smears, this method has some disadvantages. First, the parasites are cultured under harsh conditions, as the yellow color of the medium is an indication of a low pH (due to lactic acid production). Under conditions of low pH, parasites would be in a glucose-deprived state. Second, the method requires a skilled technician to detect the subtle color changes. Third, the method can only detect parasitemias in excess of 0.5%(Kirkman et al. 1996). A parasite-specific lactate dehydrogenase assay was first described by Makler et al.(1993). The pLDH assay is specific for the malarial parasite enzyme and does not detect erythrocyte LDH. It is based on the principle that malaria LDH can utilize 3-acetylpyridine NAD (APAD) as an NAD analogue, whereas erythrocyte LDH cannot (Makler and Hinrichs 1993). The assay reagents, nitroblue tetrozolium (NBT) and phenylethyl sulfate (PES), were obtained from Sigma (St. Louis, MO). The Malstat reagent was obtained from Flow, Inc.(Portland, OR). To perform the pLDH assay, 20 μl of infected erythrocytes (1% hematocrit) in each well of a 96-well plate was mixed with 100 μl of Malstat reagent, 10 μl of 1 mg/ml NBT, and 10 μl of 0.1 mg/ml PES for 30 min at 20 C. The reaction was stopped by the addition of 100 μl of 5% acetic acid to each well. The absorbance at 650 nm (A650) was read on a plate reader. The A650 has been shown to be proportional to the parasitemia detected by Giemsa-stained blood smears (Makler et al. 1993). To test the utility of the pLDH assay for the identification of malarial clones, P. falciparum (pyrimethamine-sensitive strain 3D7) were transfected by an adaptation of the method of Wu et al.(1995), with a DNA construct containing the Toxoplasma gondii dihydrofolate reductase/thymidylate synthase gene flanked by regulatory regions from the malaria knob-associated histidine-rich protein (KAHRP)(Lanzer et al. 1992). The cells were grown in the presence of 15 ng/ml pyrimethamine for 5 weeks, after which drugresistant parasites were isolated. These parasites were diluted and transferred to 96-well microtiter plates that were maintained in a low-oxygen atmosphere (1% O2, 5% CO2, 94% N2) using a Modular Chamber Incubator (Vanguard, Neptune, NJ). The medium was changed after 1 week. After another week (14 days postseeding), the pLDH assay was performed on each sample in the microtiter plates. Some of the wells were weakly positive (A650∼ 0.1). The medium was changed and the cells …