Old Matthew O, Martinez Claudia V, Kwock Douglas, Garcia Joaquin, Martin Gladys, Chan Christina, Maldonado Yvonne A
Department of Pediatrics, School of Medicine, Stanford University, MC 5208, 300 Pasteur Drive, Stanford, CA 94305, USA.
J Virol Methods. 2003 Jun 30;110(2):193-200. doi: 10.1016/s0166-0934(03)00133-2.
To permit rapid and efficient detection of Sabin poliovirus type 3 from human fecal samples, we developed a guanidine thiocyanate (GuSCN) extraction and reverse transcriptase polymerase chain reaction (RT-PCR) method. Using 10-fold serial dilutions from stock Sabin-Leon 12 a1b poliovirus type 3 at 10(7) TCID(50) per 0.1 ml, genome was detected to a dilution of 10(3) TCID(50) per 0.1 ml. A total of 40 archived fecal samples were examined using this GuSCN extraction method followed by RT-PCR. Fourteen of 20 poliovirus type 3 tissue culture-positive specimens (70%) and two of 20 tissue culture-negative specimens (10%) were detected by GuSCN extraction and RT-PCR. All positive and negative extraction and RT-PCR controls were identified accurately. This GuSCN extraction and RT-PCR technique is rapid, inexpensive, and can be readily adapted to identify genome sequences of other enterovirus types in large numbers of fecal samples. Moreover, the GuSCN technique extracts viral RNA directly from fecal samples, allowing observation of in vivo alterations of genome sequences. Further studies are underway to examine the development of revertant point mutations in the Sabin poliovirus type 3 genome following oral administration of trivalent Sabin Oral Poliovirus Vaccine to humans.
为了能够快速、高效地从人类粪便样本中检测出3型萨宾脊髓灰质炎病毒，我们开发了一种硫氰酸胍（GuSCN）提取及逆转录聚合酶链反应（RT-PCR）方法。使用每0.1 ml含10(7) TCID(50)的萨宾-莱昂12 a1b 3型脊髓灰质炎病毒原液进行10倍系列稀释，基因组在稀释至每0.1 ml含10(3) TCID(50)时仍能被检测到。使用这种GuSCN提取方法并结合RT-PCR对总共40份存档粪便样本进行了检测。在20份3型脊髓灰质炎病毒组织培养阳性标本中，有14份（70%）通过GuSCN提取及RT-PCR检测到，在20份组织培养阴性标本中有2份（10%）检测到。所有阳性和阴性提取及RT-PCR对照均被准确鉴定。这种GuSCN提取及RT-PCR技术快速、廉价，并且能够很容易地应用于大量粪便样本中其他肠道病毒类型基因组序列的鉴定。此外，GuSCN技术可直接从粪便样本中提取病毒RNA，从而能够观察基因组序列在体内的变化。目前正在进行进一步研究，以检测口服三价萨宾口服脊髓灰质炎疫苗后3型萨宾脊髓灰质炎病毒基因组中回复点突变的发生情况。
