DEVELOPMENT OF MOLECULAR BIOLOGICAL METHODS TO VACCINE AND CELL SUBSTRATE SAFETY

本项目的目标是应用和发展分子生物学。 可以确保疫苗和细胞底物不含 病毒，特别是逆转录病毒污染。 最近发表的三种敏感的检测方法 逆转录酶(RT)至少是一百万倍以上 比传统RT检测更灵敏的方法以及使用其中一种方法的发现 某些疫苗具有可检测到的RT活性水平的检测 因此，有必要让CBER开展研究以解决这一问题。这个 三种分析依赖于相同的原理：已知序列的RNA是 在RT反应中用作具有寡脱氧核苷酸引物的模板； 因为RNA的序列是已知的，所以cdna产物可以 通过聚合酶链式反应(PCR)扩增； 通过多种方法进行检测。这三种检测方法使用不同的RNA 模板：雀麦花叶病毒(BMV)基因组、MS2基因组和体外 脑心肌炎病毒(EMCV)合成产物。虽然 这些检测被不同地命名为PERT(产品增强RT)或 AMP-RT，我们选择以PBRT的通用名称来称呼它们 基于聚合酶链式反应的RT。 我们在CBER建立了三种检测方法，以比较它们的 敏感性、特异性和重复性。我们发现， MS2试验(PERT)的敏感性约为BMV的10-50倍 该方法比Amp-RT的灵敏度高10~100倍 化验。通过将BMV检测的模板-引物水平提高到 与PERT法相比，这两种方法变得同样敏感。在……里面 此外，我们还设计了可与第三方配合使用的底漆 商业上可获得的RNA、TMV，使用该模板的检测是 同样敏感。该分析的固有背景信号是 通过在添加RNase之前加入RNase消化来有效消除 步骤，并使用缺乏RT活性的耐热聚合酶。 由于PBRT分析的高度敏感性，细胞提取液产生 这是一个积极的信号，许多依赖DNA的DNA聚合酶也是如此。推理 真正的RT~S将具有使酶能够 复制RNA，我们设计了位于次要区域周围的引物 结构，这就需要制作一个更长的cdna。有了这个 改进的检测方法，细胞提取液产生的信号被减弱 几个测井记录，没有明显的灵敏度损失。重要的是 所有已知类别的逆转录病毒得分均为阳性。最近，我们有 使PBRT分析与实时定量系统一起使用， TaqMan系统，用于Perkin-Elmer 7700系统。这 改良的PBRT法，即TM-PERT法，在至少6个数量级上是线性的 与原始的PBRT检测方法一样灵敏。我们是 目前正在尝试修改这一检测方法，使其具有选择性 逆转录病毒。

The goal of this project is to apply and develop molecular biological methods that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination. The recent publications of three sensitive methods for the detection of reverse transcriptase (RT) that are at least a million-fold more sensitive than conventional RT assays and the finding using one of these assays that certain vaccines had detectable levels of RT activity necessitated that CBER undertake research to address this issue. The three assays depend on the same principle: an RNA of known sequence is used as a template with an oligodeoxynucleotide primer in an RT reaction; because the sequence of the RNA is known, the cDNA product can be amplified by the polymerase chain reaction (PCR); the PCR product can be detected by a number of methods. The three assays use different RNA templates: brome mosaic virus (BMV) genome, MS2 genome, and an in vitro synthesized product from encephalomyocarditis virus (EMCV). Although the assays were variously termed PERT (for product-enhanced RT) or Amp-RT, we have chosen to call them by the generic name of PBRT for PCR-based RT. We set up the three assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. We found that the MS2 assay (PERT) was about 10 to 50 times more sensitive than the BMV assay, which was about 10 to 100 fold more sensitive than the Amp-RT assay. By increasing the template-primer levels of the BMV assay to those of the PERT assay, the two assays became equally sensitive. In addition, we designed primers to be used with a third commercially-available RNA, TMV, and the assay with this template was similarly sensitive. The inherent background signal of the assay was effectively eliminated by incorporating an RNase digestion prior to the PCR step and using a thermostable polymerase lacking RT activity and. Because of the high sensitivity of the PBRT assay, cell extracts produce a positive signal as do many DNA-dependent DNA polymerases. Reasoning that genuine RT~s would have properties that would enable the enzyme to copy RNA, we designed primers that were around regions of secondary structure and that required that a longer cDNA had to be made. With this modified assay, the signal produced by the cell extract was diminished several logs without significant loss in sensitivity. Importantly, retroviruses from all known classes scored positive. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified PBRT assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original PBRT assays. We are currently trying to modify this assay to make it selective for retroviruses.