Hale A D, Green J, Brown D W
Enteric and Respiratory Virus Laboratory, Central Public Health Laboratory, London, UK.
J Virol Methods. 1996 Apr 5;57(2):195-201. doi: 10.1016/0166-0934(95)01966-9.
Four methods for extraction of the RNA genome of small round structured viruses (SRSVs) from faecal specimens by reverse transcription polymerase chain reaction (RT-PCR) were evaluated. The efficiency of recovery of viral RNA and removal of amplification inhibitors were compared. RNA extraction using the metal chelating agent Chelex-100 and Sephadex G200 column chromatography were the most sensitive, detecting a 10(-4) dilution of a known SRSV positive specimen. Guanidinium thiocyanate (GTC) with adsorption of viral RNA onto silica was 10-fold less sensitive. The fourth method, based on PEG precipitation followed by phenol/chloroform extraction with the addition of the detergent cetyltrimethylammonium bromide (CTAB), only detected a 1 in 10 dilution of the positive specimen. The CTAB method was 2- to 50-fold less sensitive than the GTC/silica method when dilution series of three further SRSV positive specimens were tested. Thirty-six SRSV negative faecal specimens were spiked with virus and RT-PCR performed following RNA extraction by each of the four techniques in order to assess the ability of these methods to remove amplification inhibitors. The GTC/silica method successfully removed inhibitors from all samples whereas partial or complete inhibition was observed in seven (19%) specimens following extraction by the CTAB method, 17 (47%) by the Sephadex method, and 20 (56%) by the Chelex method. We conclude that, of these four methods, the GTC/silica method is the most appropriate for the extraction of viral RNA from faecal samples prior to RT-PCR for detecting SRSVs.
对通过逆转录聚合酶链反应（RT-PCR）从粪便标本中提取小圆结构病毒（SRSV）RNA基因组的四种方法进行了评估。比较了病毒RNA回收效率和扩增抑制剂去除情况。使用金属螯合剂Chelex-100和葡聚糖凝胶G200柱色谱法提取RNA最为灵敏，可检测到已知SRSV阳性标本10⁻⁴的稀释度。将硫氰酸胍（GTC）与病毒RNA吸附到硅胶上的方法灵敏度低10倍。第四种方法是基于聚乙二醇沉淀，随后用苯酚/氯仿提取并添加去污剂十六烷基三甲基溴化铵（CTAB），仅能检测到阳性标本1/10的稀释度。当对另外三个SRSV阳性标本的稀释系列进行测试时，CTAB法的灵敏度比GTC/硅胶法低2至50倍。用病毒对36份SRSV阴性粪便标本进行加样，并在通过四种技术中的每一种进行RNA提取后进行RT-PCR，以评估这些方法去除扩增抑制剂的能力。GTC/硅胶法成功去除了所有样品中的抑制剂，而CTAB法提取后在7份（19%）标本中观察到部分或完全抑制，葡聚糖凝胶法为17份（47%），Chelex法为20份（56%）。我们得出结论，在这四种方法中，GTC/硅胶法最适合在RT-PCR检测SRSV之前从粪便样本中提取病毒RNA。
