DETECTION OF HEPATITIS C VIRUS RNA BY PCR IN PLASMA DERIVATIVES

血浆衍生物中丙型肝炎病毒 RNA 的 PCR 检测

• 批准号: 3792651
• 负责人: M W YU
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH HEMATOLOGY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792651
• 关键词: Pan; blood fractionation; coagulation factor IX; communicable disease transmission; hepatitis C virus; human tissue; immunoglobulin G; pancreatic ribonuclease; plasma; polymerase chain reaction; virus RNA

Because the sensitivity of our "nested" PCR method is at least equivalent to the chimpanzee model, we proceeded to use the method to assay HCV RNA in plasma derivatives such as IGIV, IG, and AHF. An ultracentrifugal method (50,000 rpm for 3 h with a 70 Ti rotor, ca. 180,000 x g) was developed to concentrate "virus" in the pellet while at least 90% of the protein remained in the supernatant. We assayed seven experimental lots, produced by 7 manufacturers from Source Plasma (2,887 units) that had been screened for anti-HCV (anti-c100-3). HCV RNA was detected in one of these experimental lots, even through there was no evidence of hepatitis C transmission when this lot, along with the other six PCR-negative lots, was infused into 3 chimpanzees. We also analyzed 32 routine lots of IGIV (prepared from plasma not screened for anti-HCV) from the 7 manufacturers. HCV RNA was detected in four of these lots, all from one manufacturer (the same manufacturer whose experimental IGIV lot, produced from screened plasma, was positive). These five positive lots were determined by limiting dilution analysis to have from 10 to 250 PCR U/g IgG. It is of interest that 4 IGIV lots produced by this manufacturer from recovered plasma was negative for HCV RNA, whereas 4 out of 5 lots derived from Source Plasma were positive. One lot of VZIG prepared from recovered plasma by the same manufacturer was also negative. This may be due to the lower anti-HCV reactive rate, hence, the lower viral load, in recovered plasma. Two intramuscular IG lots and two older AHF lots were assayed and found to contain 30 and 260 PCR U/g IgG, and 0.1 and 4 PCR U/IU FVIII, respectively. One lot of Coagulation Factor IX (Human) (purified, heated in heptane) was assayed and tested negative. Preliminary data obtained from studies utilizing Rnase A digestion suggest that the HCV RNA presented in immune globulins may be as a "naked" RNA rather than intact virus; this may explain how viral RNA can be present in some immune globulin products without transmitting hepatitis C to recipients. Detection and quantitation of HCV RNA and its digestibility by Rnase A in plasma fractionation products will be continued.

因为我们的“巢式”PCR方法的灵敏度至少相当于 在黑猩猩模型中，我们继续使用该方法检测 等离子体衍生物，如IGIV、IG和AHF。 超离心法 (50，OOOrpm搅拌3小时，使用70 Ti转子，约. 180，000 x g）， 在沉淀物中浓缩“病毒”，而至少90%的蛋白质 保留在上清液中。 我们分析了七个实验批次， 来源血浆（2，887单位）的7家制造商进行了筛选 抗HCV（抗c100 -3）。 在其中一个组织中检测到HCV RNA， 实验批次，即使没有丙型肝炎的证据， 当该批次与其他6个PCR阴性批次沿着 注入三只黑猩猩体内 我们还分析了32批常规IGIV （由未筛选抗HCV的血浆制备）。 在这些批次中的四个批次中检测到HCV RNA，所有批次均来自一个制造商（ 同一生产商，其实验IGIV批次由筛选的 血浆，为阳性）。 这5个阳性批次的测定方法如下： 有限稀释分析以具有10至250 PCR U/g IgG。 具有 令人感兴趣是该制造商从回收的IGIV中生产的4个批次 血浆中HCV RNA为阴性，而5批中有4批来自 源血浆呈阳性。 用回收的VZIG制备一批VZIG 同一厂家的血浆也呈阴性。 这可能是由于 较低的抗-HCV反应率，因此，较低的病毒载量，在恢复 等离子体 对两个肌内IG批次和两个较旧的AHF批次进行了分析， 发现含有30和260 PCR U/g IgG，以及0.1和4 PCR U/IU FVIII， 分别 一批凝血因子IX（人）（纯化，加热 在庚烷中）进行测定并测试为阴性。 获得的初步数据 利用RNase A消化的研究表明， 免疫球蛋白中的RNA可能是“裸露的”RNA，而不是完整的病毒; 可以解释病毒RNA如何存在于某些免疫球蛋白产品中 而不会将丙型肝炎传染给接受者。 检测和定量 血浆中HCV RNA的分离及其RNA酶A消化率 产品将继续。