Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples
Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples
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DOI：
10.1128/aem.03082-14
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发表时间：
2015-03-01
影响因子：
4.4
通讯作者：
Furumai, Hiroaki
中科院分区：
文献类型：
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作者：
Hata, Akihiko;Katayama, Hiroyuki;Furumai, Hiroaki
从水样中的逆转录（RT）-PCR为基础的病毒检测偶尔会受到有机物质，在病毒浓缩过程中被共浓缩。为了表征这些有机物质，含有市售腐殖酸的样品，这是已知的抑制RT-PCR，和河水样品进行吸附洗脱为基础的病毒浓度使用负电性膜。在这项研究中，样品之前，期间，和之后的浓度进行了分析的有机性质和病毒检测效率。三种腐殖酸溶液中有两种导致RT-定量PCR（qPCR）抑制，导致加标脊髓灰质炎病毒低估>3-log（10）-单位。两种溶液中60%以上的有机物在浓缩液中回收，而未抑制溶液中60%以上的有机物在浓缩过程中损失。河水浓缩物也引起RT-qPCR的抑制。在病毒浓缩程序后，河水样品中的有机物浓度增加了2.3至3.9倍。抑制性样品含有10- 100-kDa大小范围内的有机组分，其被怀疑是RT-PCR抑制剂。根据激发发射矩阵，腐殖酸样和蛋白质样馏分也回收河水浓缩物，但这些馏分似乎并不影响病毒检测。我们的研究结果表明，详细的有机分析是有效的表征抑制物质。
Reverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log(10)-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances.