RIBONUCLEOTIDE VANADYL COMPLEXES INHIBIT POLYMERASE CHAIN-REACTION
RIBONUCLEOTIDE VANADYL COMPLEXES INHIBIT POLYMERASE CHAIN-REACTION
复制标题
DOI：
10.1093/nar/21.11.2777
复制
发表时间：
1993-06-11
影响因子：
14.9
通讯作者：
DAVIS, GL
中科院分区：
文献类型：
--
作者：
LAU, JYN;QIAN, KP;DAVIS, GL
核糖核苷酸钒基复合物（RVC）通常用于抑制RNA酶活性（1）。在从福尔马林固定的石蜡组织中分离RNA的过程中，在长时间的蛋白水解消化过程中，它被用来抑制RNA酶。基于这种方法，成功地从福尔马林固定的肝组织中分离出丙型肝炎病毒（HCV）RNA，并通过逆转录巢式聚合酶链反应（RT-nPCR）进行检测（2）。然而，使用类似的方案，我们无法检测HCV序列，并发现微量的RVC可以抑制PCR。对10例HCV抗体和HCV RNA阳性患者的福尔马林固定石蜡包埋切片进行检测。通过标准二甲苯和乙醇洗涤除去石蜡。然后在20 mM RVC（Sigma，圣刘易斯，MO）存在下，用蛋白酶K在42 ℃消化组织4-5天.然后通过酚-氯仿提取法提取RNA，并根据先前描述的方案（3，4）对RNA进行RT-nPCR。未检测到扩增子。从例慢性HCV感染患者血清中提取的已知阳性HCV RNA加入到提取混合物中，同样没有检测到扩增子，表明RVC抑制RT或PCR。为了直接阐明RVC对PCR的抑制作用，将HCV cDNA用作模板，并将一系列浓度的RVC（0、0.04、0.4、2、4、8 mM）加入到PCR反应混合物中。发现0.4mM或更高浓度的RVC始终抑制PCR反应，并且0.04mM的RVC没有可观察到的抑制效果。与Sigma Chemical公司讨论了该问题，并将另一批RVC发送给我们进行重新检测，在平行浓度的RVC中始终观察到相似的抑制活性。然后，我们根据先前描述的方案测试了另一种DNA模板，即B型肝炎病毒DNA（5）。同样，PCR反应被0.4 mM RVC抑制。
Ribonucleotide vanadyl complexes (RVC) are commonly used to inhibit RNase activity (1). It has been used to inhibit RNases during the isolation of RNA from formalin fixedparaffin tissue during prolonged proteolytic digestion. Based on this approach, hepatitis C virus (HCV) RNA was successfully isolated from formalin-fixed liver tissue and detected by reverse-transcription nested polymerase chain reaction (RT-nPCR)(2). However, using a similar protocol, we were unable to detect HCV sequences and found that trace amounts of RVC can inhibit PCR. Formalin-fixed paraffin embedded sections from 10 patients seropositive for antibody to HCV and HCV RNAwere tested. Paraffin was removed by standard xylene and ethanol washes. The tissue was then digested using proteinase K at 42 C for 4-5 days in the presence of 20 mM RVC (Sigma, St Louis, MO). RNA was then extractedby phenol-chloroform extraction and the RNA was subjected to RT-nPCR according to a previously described protocol (3, 4). No amplicon was detected. Known positive HCV RNA extracted from sera ofpatients withchronic HCV infection was then added to the extraction mixture and again no amplicon was detected, suggesting that RVC inhibits either RT or PCR. To elucidate directly the inhibitory effect of RVC on PCR, HCV cDNA was used as a template and a range of concentrations of RVC (0, 0.04, 0.4, 2, 4, 8 mM) was added to the PCR reaction mixture. It was found that 0.4 mM or higher concentrations of RVC consistently inhibited PCR reaction and 0.04 mM of RVC had no observable inhibitory effect. The problem was discussed with Sigma Chemical company and another batch of RVC was sent to us for re-testing and similar inhibitory activity was consistently observed with parallel concentrations of RVC. We then tested another DNA template, hepatitis B virus DNA, according to a previously described protocol (5). Again, the PCR reaction was inhibited by 0.4 mM RVC.