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首页> 外文期刊>Applied and Environmental Microbiology >Electropositive Filter Membrane as an Alternative for the Elimination of PCR Inhibitors from Sewage and Water Samples
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Electropositive Filter Membrane as an Alternative for the Elimination of PCR Inhibitors from Sewage and Water Samples

机译：用正电滤膜替代污水和水样中的PCR抑制剂

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PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natura sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eight samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additional sample purification methods.
机译：PCR是检测环境样品中低浓度肠病毒的最佳方法。但是，这些样品中存在的某些有机和无机化合物会干扰反应。这些物质中许多也具有细胞毒性。除氟戊烷处理外使用的ZP60S滤膜对于病毒浓缩和同时消除环境样品的细胞毒性非常有效。在这项研究中，这两种方法均被用来促进从污水和污水污染的小溪中消除逆转录酶PCR（RT-PCR）抑制剂。分别对样品进行以下操作：通过正电滤膜（ZP60S）过滤，使用Vertrel XF进行有机萃取，以及通过ZP60S过滤，然后进行有机萃取。之后，将等分试样用轮状病毒SA-11 RNA进行实验接种，并进行RT-semines PCR扩增VP7基因。结果表明，ZP60S膜可有效去除水样中的RT-PCR抑制剂。样品处理方法还应用于31个自然污水和小溪水样品中，用于检测天然的轮状病毒。双链半巢式PCR用于快速检测四种轮状病毒基因对（G1至G4）。八个样品（占25.8％）为阳性，在23个样品中（74.2％）未检测到轮状病毒序列。通过直接免疫过氧化物酶法证实了结果。总之，使用正电滤膜可消除可能干扰RT-PCR的物质，从​​而避免了其他样品纯化方法。

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