Organic substances interfere with reverse transcription-quantitative PCR-based virus detection in water samples

Area of Science:

• Environmental science
• Molecular biology
• Analytical chemistry

Background:

• Reverse transcription polymerase chain reaction (RT-PCR) is crucial for detecting viruses in water.
• Organic substances co-concentrated during sample preparation can inhibit RT-PCR, affecting detection accuracy.

Purpose of the Study:

• To characterize organic substances that inhibit RT-PCR in water samples.
• To understand the role of organic matter in RT-PCR inhibition during virus concentration.

Main Methods:

• Used humic acid and river water samples for adsorption-elution virus concentration via electronegative membranes.
• Analyzed organic properties and virus detection efficiencies before, during, and after concentration.
• Utilized size fractionation and excitation-emission matrices to identify inhibitory organic compounds.

Main Results:

• Two of three humic acid solutions inhibited RT-quantitative PCR (qPCR), causing significant underestimation of spiked poliovirus.
• Over 60% of inhibitory organics from humic acid solutions were recovered in the concentrate.
• River water concentrates showed increased organic concentrations and inhibited RT-qPCR, with inhibitory fractions in the 10- to 100-kDa range.

Conclusions:

• Organic substances, particularly those in the 10- to 100-kDa range, are responsible for RT-PCR inhibition in concentrated water samples.
• Detailed organic analysis is effective for identifying RT-PCR inhibitors in environmental samples.
• Understanding organic interference is key to reliable virus detection in water quality assessments.