Poddar S K
Department of Pediatrics, University of California at San Diego, La Jolla 92093-0808, USA.
J Virol Methods. 1999 Sep;82(1):19-26. doi: 10.1016/s0166-0934(99)00074-9.
The polymerase chain reaction (PCR) and a molecular beacon probe were used for the detection of Adenovirus. A 307 bp DNA fragment from a conserved region of the hexon gene was amplified. The specific molecular beacon was characterized with respect to its efficiency of quenching, and signal to noise ratio by spectrofluorometric analysis of its hybridization with virus specific complementary single stranded oligonucleotide target. Amplification was carried out in the presence of the molecular beacon probe, and the amplified target was detected by measurement of fluorescence signal in the post PCR sample. Separately, a 32P-labeled linear probe (having the same sequence as that of molecular beacon probe) was liquid-phase hybridized with the product of PCR performed in the absence of the molecular beacon. The virus specific target was then detected by electrophoresis of the hybridized product in a nondenaturing polyacrylamide gel and subsequent autoradiographic analysis. The detection limit of adenovirus by PCR in the presence of the molecular beacon probe was found to be similar to that obtained by labeled linear probe hybridization following PCR.
采用聚合酶链反应（PCR）和分子信标探针检测腺病毒。扩增了六邻体基因保守区的一段307 bp DNA片段。通过对其与病毒特异性互补单链寡核苷酸靶标的杂交进行荧光光谱分析，对特异性分子信标进行了淬灭效率和信噪比的表征。在分子信标探针存在的情况下进行扩增，并通过测量PCR后样品中的荧光信号来检测扩增的靶标。另外，将一个32P标记的线性探针（与分子信标探针序列相同）与在没有分子信标的情况下进行PCR的产物进行液相杂交。然后通过在非变性聚丙烯酰胺凝胶中对杂交产物进行电泳并随后进行放射自显影分析来检测病毒特异性靶标。发现在分子信标探针存在的情况下，PCR检测腺病毒的检测限与PCR后标记线性探针杂交获得的检测限相似。
