Purification and characterization of PCR-inhibitory components in blood cells
Purification and characterization of PCR-inhibitory components in blood cells
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DOI：
10.1128/jcm.39.2.485-493.2001
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发表时间：
2001-02-01
影响因子：
9.4
通讯作者：
Rådström, P
中科院分区：
文献类型：
--
作者：
Abu al-Soud, W;Rådström, P
在最近的一项研究中，人血浆中的免疫球蛋白G被确定为诊断PCR的主要抑制物(W.Abu Al-Soud，L.J.Jonsson和P.Radstrom)。J·克莱恩。微生物。38：345-350,2000)。在这项研究中，使用尺寸排除和阴离子交换层析程序纯化了人体血细胞中的两种主要的聚合酶链式反应抑制物。根据N端氨基酸序列测定和纯化多肽的电泳法分析，血红蛋白和乳铁蛋白分别为红细胞和白细胞中的聚合酶抑制物组分。当将不同浓度的血红蛋白或乳铁蛋白加入含有10种不同耐热DNA聚合酶的25 MUL和1 ng单核细胞增生性李斯特菌DNA作为模板DNA的PCR混合物中时，AmpliTaq Gold、Pwo和Ultma在小于或等于1.3杯血红蛋白和小于或等于25 ng乳铁蛋白的存在下被抑制，而rTth和Tli被发现能抵抗至少100杯血红蛋白的抑制。此外，使用LightCycler仪器研究了血样或血红蛋白降解产物中存在的七种低分子质量抑制物对rTth实时DNA合成的定量影响。使用了基于单链聚(DA)模板的反应系统，其中寡聚(DT)引物3‘端退火。结果发现，每英里加入0.25-0.1毫克胆汁、2.5 mM CaCl2、0.25 mM EDTA、5微米FeCl2和0.01IU肝素，荧光强度分别降低到约76%、70%、46%、17%和51%。最后，研究了9种扩增促进剂在血红蛋白和乳铁蛋白存在下的作用。牛血清白蛋白(BSA)是最有效的扩增促进剂，因此添加0.4%的牛血清白蛋白使AmpliTaq Gold能够在20而不是1杯血红蛋白和500而不是5 ng乳铁蛋白的存在下扩增DNA。在AmpliTaq Gold的反应混合物中加入0.02%(wt/Vol)的单链DNA结合蛋白gp32，也可以减弱血红蛋白和乳铁蛋白的抑制作用。
In a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jonsson, and P. Radstrom. J. Clin. Microbiol. 38:345-350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 mul containing 10 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, AmpliTaq Gold, Pwo, and Ultma were inhibited in the presence of less than or equal to1.3 mug of hemoglobin and less than or equal to 25 ng of lactoferrin, while rTth and Tli were found to resist inhibition of at least 100 mug of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis of rTth using the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3' end was used. It was found that the addition of 0.25 to 0.1 mg of bile per mi, 2.5 mM CaCl2, 0.25 mM EDTA, 5 muM FeCl2, and 0.01 IU of heparin per mi reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% wt/vol) BSA allowed AmpliTaq Gold to amplify DNA in the presence of 20 instead of 1 mug of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaq Gold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.