Ijzerman M M, Dahling D R, Fout G S
Biohazard Assessment Research Branch, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USA.
J Virol Methods. 1997 Jan;63(1-2):145-53. doi: 10.1016/s0166-0934(96)02123-4.
A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR). Environmental inhibitors, concentrated along with viruses during water sample processing, are removed by the method through a series of steps that includes dialysis, solvent extraction, ultrafiltration and glass purification. The method was tested by spiking sodium phosphate with poliovirus type 1 with or without humic or fulvic acids and then measuring virus recovery by plaque assay and RT-PCR. Results of the study indicated that (i) 90% of the spiked virus could be recovered from samples at the end of the ultrafiltration step, (ii) virus was detected in the final eluate of samples containing as much as 0.5 mg of humic acid or 5.0 mg of fulvic acid, and (iii) as little as 0.06 plaque forming units (PFU) was detectable per RT-PCR reaction. These results indicate that the described purification method along with RT-PCR is a feasible approach for detecting waterborne human enteric viruses in the presence of interfering substances.
开发了一种方法，用于在使用逆转录聚合酶链反应（RT-PCR）检测人类肠道病毒之前，从样品浓缩物中去除环境抑制剂。在水样处理过程中与病毒一起浓缩的环境抑制剂，通过该方法的一系列步骤被去除，这些步骤包括透析、溶剂萃取、超滤和玻璃纯化。通过在含或不含腐殖酸或富里酸的情况下，向磷酸钠中加入1型脊髓灰质炎病毒，然后通过噬斑测定和RT-PCR测量病毒回收率，对该方法进行了测试。研究结果表明：（i）在超滤步骤结束时，90%的加标病毒可从样品中回收；（ii）在含有高达0.5毫克腐殖酸或5.0毫克富里酸的样品的最终洗脱液中检测到病毒；（iii）每个RT-PCR反应可检测到低至0.06个噬斑形成单位（PFU）。这些结果表明，所述纯化方法与RT-PCR相结合，是在存在干扰物质的情况下检测水源性人类肠道病毒的一种可行方法。
