Design of an antisense reverse-transcriptase-polymerase chain reaction primer efficient for all hepatitis C virus genotypes:: Comparison of its performance vs a commercial primer
Design of an antisense reverse-transcriptase-polymerase chain reaction primer efficient for all hepatitis C virus genotypes:: Comparison of its performance vs a commercial primer
复制标题
DOI：
10.1016/s0003-2697(02)00703-0
复制
发表时间：
2003-04-15
影响因子：
2.9
通讯作者：
Gonz치lez, YJG
中科院分区：
文献类型：
--
作者：
Gonzalez-Perez, I;Vina-Rodriguez, A;Gonz치lez, YJG
材料与方法srna提取、rt -巢式PCR、扩增DNA检测。我们采用Umelosa HCV定性试验[4]（古巴哈瓦那免疫分析中心），这是一种rt -巢式PCR方法，用于检测人血清或血浆中的HCVRNA。测试分三步进行：(a)使用Chomckzynsky和Sacchi[5]的苯酚-氯仿法的变体提取病毒RNA，(b)单管逆转录目标RNA和PCR（外部引物HCV011和HCV012或HCV012N），再加上第二轮扩增（内部引物HCV013和HCV014）， (c)将扩增的DNA在包被互补探针的固相上杂交。在SUMA PR-521平板阅读器（免疫分析中心）上进行最终荧光检测，采用共轭链亲和-磷酸酶碱性（免疫分析中心）和底物4-甲基伞形酰基磷酸（Koch Light Ltd.）。哈弗希尔，萨福克，英格兰)。所有PCR产物均在杂交步骤中重复检测。
Materials and methodsRNA extraction, RT-nested PCR, and detection of the amplified DNA. We employed the Umelosa HCV Cualitativo test [4](ImmunoAssay Center, Havana, Cuba), an RT-nested PCR assay for the detection of HCVRNA in human serum or plasma. The test was performed in three steps:(a) extraction of viral RNA using a variant of the phenol-chloroform method of Chomckzynsky and Sacchi [5],(b) single-tube reverse transcription of target RNA and PCR (outer primers HCV011 and HCV012 or HCV012N), coupled with a second round of amplification (inner primers HCV013 and HCV014), and (c) hybridization of the amplified DNA on a solid phase coated with a complementary probe, for final fluorescent detection on a SUMA PR-521 plate reader (ImmunoAssay Center), employing a conjugate streptavidine-phosphatase alkaline (Immuno-Assay Center) and substrate 4-methylumbelliferylphosphate (Koch Light Ltd. Haverhill, Suffolk, England). All PCR products were detected in duplicate in the hybridization step.