Wolff C, Schlüter K, Prohaska W, Kleesiek K
Herzzentrum Nordrhein-Westfalen, Universitätsklinik, Ruhr-Universität Bochum.
Eur J Clin Chem Clin Biochem. 1992 Nov;30(11):717-27. doi: 10.1515/cclm.1992.30.11.717.
The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.
将RNA先逆转录为cDNA，然后进行聚合酶链反应，用于检测不同来源的人血清样本中的丙型肝炎病毒RNA。为消除假阴性结果，对以下步骤进行了优化：RNA提取、逆转录和寡核苷酸引物选择。我们比较了使用胍盐/去污剂以及蛋白酶K消化/苯酚提取的不同RNA提取方法，并测试了用聚乙二醇沉淀和超速离心富集病毒颗粒的效果。用胍盐/去污剂提取RNA是最有效的方法。对单个样本进行超速离心并不能提高丙型肝炎病毒RNA的检测率。聚乙二醇沉淀效果不佳。与莫洛尼鼠白血病病毒逆转录酶相比，重组热稳定逆转录酶从较少的样本中产生cDNA。来自丙型肝炎病毒基因组5'端非编码区的巢式寡核苷酸引物比来自编码区的引物能从更多样本中扩增出cDNA。对36份抗丙型肝炎病毒抗体阳性样本进行了检测；巢式引物（核苷酸6至327和15至288）产生了21个扩增产物，而来自编码区的引物分别产生了16个扩增产物（核苷酸4684 - 5276）和5个扩增产物（核苷酸5166 - 5270）。最有效的步骤组合是用胍盐溶液提取RNA，用莫洛尼鼠白血病病毒逆转录酶进行逆转录，并用来自丙型肝炎病毒基因组5'端非编码区的引物进行巢式聚合酶链反应。其他组合产生了更多假阴性结果。三组不同的抗丙型肝炎病毒抗体阳性个体具有明显不同的病毒血症模式：在抗丙型肝炎病毒抗体阳性的献血者中，仅10%的血清中检测到丙型肝炎病毒RNA，但在有临床症状的丙型肝炎抗丙型肝炎病毒抗体阳性患者中，这一比例为90%，在过去接受过未进行病毒灭活的血浆制品的抗丙型肝炎病毒抗体阳性血友病患者中，这一比例也为90%。在450名血清丙氨酸氨基转移酶催化浓度升高的抗丙型肝炎病毒抗体阴性献血者的血清中未检测到丙型肝炎病毒RNA。
