Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens
Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens
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DOI：
10.1016/0166-0934(95)01966-9
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发表时间：
1996-04-05
影响因子：
3.1
通讯作者：
Brown, DWG
中科院分区：
文献类型：
--
作者：
Hale, AD;Green, J;Brown, DWG
对4种从粪便标本中提取小圆结构病毒（SRSV）RNA基因组的逆转录聚合酶链反应（RT-PCR）方法进行了评价。比较病毒RNA的回收效率和扩增抑制剂的去除效率。用金属螯合剂Chelex-100和Sephadex G200柱层析法提取RNA是最灵敏的，可检测到已知SRSV阳性标本的10（-4）稀释度。硫氰酸胍（GTC）与病毒RNA吸附到二氧化硅上的敏感性低10倍。第四种方法基于PEG沉淀，然后加入洗涤剂十六烷基三甲基溴化铵（CTAB）进行苯酚/氯仿提取，仅检测到1/10稀释的阳性标本。当对另外三份SRSV阳性标本的稀释系列进行检测时，CTAB法的灵敏度比GTC/硅胶法低2 - 50倍。向36份SRSV阴性粪便标本加标病毒，并在通过4种技术提取RNA后进行RT-PCR，以评估这些方法去除扩增抑制剂的能力。GTC/二氧化硅方法成功地从所有样品中去除抑制剂，而部分或完全抑制观察到7个（19%）标本提取后的CTAB法，17（47%）的葡聚糖凝胶法，和20（56%）的Chelex方法。我们的结论是，这四种方法中，GTC/硅胶法是最合适的提取病毒RNA的粪便样品之前，RT-PCR检测SRSVs。
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.