Parvaz P, Guichard E, Chevallier P, Ritter J, Trepo C, Sepetjan M
Laboratoire de Médecine Préventive et d'Hygiène, Faculté de Médecine Lyon Nord, France.
J Virol Methods. 1994 Apr;47(1-2):83-94. doi: 10.1016/0166-0934(94)90068-x.
To improve the sensitivity of hepatitis C virus RNA (HCV RNA) detection in serum by 'nested' polymerase chain reaction (PCR), primers belonging to 5' non-coding (5'NC) regions were used to compare the classical phenol/chloroform technique by using the proteinase K and silica gel technique with guanidinium thiocyanate. The silica gel techniques was found to be more efficient and sensitive for the extraction and purification of viral RNA from serum samples. The silica gel technique also avoids contact with hazardous volatile chemicals like phenol and chloroform and provides a better protection for viral RNA. Furthermore, the RNA detection sensitivity was greatly improved by modifying the buffer for reverse transcription and PCR. Using silica gel extraction, and the modified buffer, viral RNA was detected in 699 sera from anti-HCV second generation ELISA positive patients. These sera were distributed in second generation RIBA confirmed, indeterminate and non confirmed groups with PCR positive rates of 71.4%, 45.7% and 15.8%, respectively. Two out of 227 ELISA negative patients showed the presence of HCV RNA in serum. An association between the presence of antibodies against a determined viral peptide and viremia was not detected.
为了通过“巢式”聚合酶链反应（PCR）提高血清中丙型肝炎病毒RNA（HCV RNA）检测的灵敏度，使用属于5'非编码（5'NC）区域的引物，通过蛋白酶K和含硫氰酸胍的硅胶技术，与经典的苯酚/氯仿技术进行比较。结果发现，硅胶技术在从血清样本中提取和纯化病毒RNA方面更高效、更灵敏。硅胶技术还避免了与苯酚和氯仿等有害挥发性化学物质接触，并为病毒RNA提供了更好的保护。此外，通过修改逆转录和PCR的缓冲液，RNA检测灵敏度得到了极大提高。使用硅胶提取和改良缓冲液，在抗HCV第二代ELISA阳性患者的699份血清中检测到了病毒RNA。这些血清分布在第二代RIBA确诊、不确定和未确诊组中，PCR阳性率分别为71.4%、45.7%和15.8%。227名ELISA阴性患者中有2名血清中检测到HCV RNA。未检测到针对特定病毒肽的抗体存在与病毒血症之间的关联。
