Hsuih T C, Park Y N, Zaretsky C, Wu F, Tyagi S, Kramer F R, Sperling R, Zhang D Y
Department of Pathology, Mount Sinai Hospital, New York 10029, USA.
J Clin Microbiol. 1996 Mar;34(3):501-7. doi: 10.1128/jcm.34.3.501-507.1996.
A simple, sensitive, and specific ligation-dependent PCR (LD-PCR) method for the detection of hepatitis C virus (HCV) RNA in serum is described. The assay uses two DNA capture probes for RNA isolation and two DNA hemiprobes for subsequent PCR. Each capture probe has a 3' sequence complementary to the conserved 5' untranslated region of HCV RNA and a biotin moiety at the 5' end capable of interacting with streptavidin-coated paramagnetic beads. Each hemiprobe contains a sequence complementary to the 5' untranslated region in juxtaposition to one another and a common sequence for PCR primer binding. In guanidinium thiocyanate solutions, the capture probes and the hemiprobes form a hybrid with their target, and the hybrid can be isolated from serum by the binding of the capture probes to the paramagnetic beads in the presence of a magnetic field. The hemiprobes can then be linked to each other by incubation with T4 DNA ligase to form a full probe that serves as a template for a PCR. When serial 10-fold dilutions of synthetic HCV RNA (10(7) to 10 molecules) were tested, there was a good correlation between the amount of PCR product and the initial number of RNA molecules, with a sensitivity of 100 HCV RNA molecules per reaction. Twenty-four specimens that had been tested by either a branched DNA probe (bDNA) assay (13 specimens) or a reverse transcription PCR (RT-PCR) assay (11 specimens) were also analyzed by LD-PCR. The results showed a good correlation among LD-PCR, RT-PCR, and the bDNA assay. However, both LD-PCR and RT-PCR were more sensitive than the bDNA assay when the HCV titer was low.
本文描述了一种用于检测血清中丙型肝炎病毒（HCV）RNA的简单、灵敏且特异的连接依赖型PCR（LD-PCR）方法。该检测方法使用两种DNA捕获探针进行RNA分离，以及两种DNA半探针用于后续PCR。每个捕获探针具有与HCV RNA保守的5'非翻译区互补的3'序列，并且在5'端具有能够与链霉亲和素包被的顺磁性珠相互作用的生物素部分。每个半探针包含彼此并列的与5'非翻译区互补的序列以及用于PCR引物结合的共同序列。在硫氰酸胍溶液中，捕获探针和半探针与其靶标形成杂交体，并且在磁场存在下，通过捕获探针与顺磁性珠的结合，可以从血清中分离出杂交体。然后，通过与T4 DNA连接酶孵育，半探针可以彼此连接形成完整的探针，该探针用作PCR的模板。当对合成HCV RNA的系列10倍稀释液（10⁷至10个分子）进行检测时，PCR产物量与RNA分子初始数量之间具有良好的相关性，每个反应的灵敏度为100个HCV RNA分子。还通过LD-PCR分析了24个已通过分支DNA探针（bDNA）检测（13个样本）或逆转录PCR（RT-PCR）检测（11个样本）的标本。结果显示LD-PCR、RT-PCR和bDNA检测之间具有良好的相关性。然而，当HCV滴度较低时。LD-PCR和RT-PCR均比bDNA检测更灵敏。
