Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

Abstract

bReverse 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. Human enteric viruses are etiological agents that can cause clinical symptoms, such as diarrhea and vomiting. Feces and vomit from infected individuals contain a substantial amount of viruses that contaminate the water environment. Consumption of improperly treated drinking water or contaminated environmental water during recreational activity leads to waterborne infections (1, 2).Quantitative detection of viruses present in water has been carried out worldwide in studies on the fate of viruses in the environment and potential viral infection risk (3-5). The detection of viruses in a water sample is commonly carried out using a PCR assay following virus concentration and nucleic acid extraction steps due to its superior rapidity, specificity, and sensitivity. However, the reliability of the PCR-based assay occasionally has come into question, as substances present in original samples or additives used during sample processing interfere with the nucleic acid extraction and reverse transcription (RT)-PCR (6-8). Even though several virus concentration methods have been developed, none of them can exclude the inhibitory substances completely (9).Polyvalent cations and some organic substances, such as beef extract constituents and humic acids, are known to inhibit RT-PCR (8, 10). Often, the virus detection efficiency is determined by spiking a known amount of viruses or nucleic acids in a sample and recovering them (6, 7, 11). In the worst case, the concentrations of viruses were underestimated by...

From Lab to Lake – Evaluation of Current Molecular Methods for the Detection of Infectious Enteric Viruses in Complex Water Matrices in an Urban Area

“…Organic substances are known to interfere with this step in the processing of environmental samples and thereby can reduce the precision and robustness of the detection in direct comparison to DNA viruses like HAdV [58] which could explain the shift in the detection rates.…”

Impact of Various Humic Acids on EMA-RT-qPCR to Selectively Detect Intact Viruses in Drinking Water

“…With higher MW, humic acid tends to be more aromatic and absorptive [24,25]. The same humic acid products (Ald, Wa, and IH) were studied and found that the peak MW of Ald and Wa were between 10 and 100 kDa while this value of IH was only around 1.0 kDa [17]. Therefore, it might result in the higher recovery of Ald, Wa, and Na than that of IH in the 1st concentration step.…”

“…Previous studies reported that humic acid can be co-concentrated by membrane filtration due to its adsorptive properties [17,23], which depend on the MW [24]. With higher MW, humic acid tends to be more aromatic and absorptive [24,25].…”

“…Humic acids, composed of a heterogeneous mixture of organic compounds, are ubiquitous in water sources and persistently present in treated drinking water. They can be co-concentrated with target viruses in membrane-based methods of virus concentration, and subsequently interfere with the following detection methods [17,18]. In addition, the interferences become more severe when a large volume of water samples is concentrated.…”

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

“…Yet, the RT reactions for standard curves constructed using Sensiscript and Omniscript produced reliable Cts at target concentrations as low as 10 3 copies/µl, similar to that observed for SSIV and SSIII (except for Sensi-RH: lower limit at 10 4 copies/µl). This indicates that the lower performances observed for Sensiscript and Omniscript in the environmental spike experiment could be due to inhibition of the enzymes from co-extracted components in environmental RNA(Hata et al, 2015) and/or the presence of background RNA. The later explanation contrasts with the results obtained by Levesque-Sergerie and co-workers(Levesque-Sergerie et al, 2007) who observed a general increase in the recovered copies of a spike (i.e.…”