PCR Inhibitor Levels in Concentrates of Biosolid Samples Predicted by a New Method Based on Excitation-Emission Matrix Spectroscopy

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 ؋ 10 2 to 2.3 ؋ 10 4 cell equivalents liter ؊1 , whereas GR-corrected abundances ranged from 4.7 ؋ 10 3 to 1.6 ؋ 10 6 cell equivalents liter ؊1 . GR-corrected qPCR results were in good agreement with an independent cellbased direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. Vibrio cholerae is a waterborne bacterium found worldwide in brackish water, coastal areas, and estuarine environments (1). Although the species V. cholerae comprises more than 200 serotypes, only serotypes O1 and O139 are currently able to cause epidemic and pandemic cholera outbreaks, with more than 100,000 reported death cases per year (2). All other non-O1/non-O139 serotypes are usually associated with less-severe gastrointestinal, blood, wound, and ear infections (3). V. cholerae has been classified as a potential category B terrorism agent by the U.S. Centers for Disease Control and Prevention (4). Detection and quantification of V. cholerae, especially of serogroup O1/O139 strains in environmental samples, are still difficult tasks, and no international standard is available. Currently accepted cultivation-based methods take at least 48 h to obtain a final result (5) and severely underestimate the presence of O1/O139 serogroups, due to the fact that these strains predominantly enter a viable but nonculturable (VBNC) state once released from the human intestine into the environment (6, 7). Alternatively, fluorescence in situ hybridization (FISH) and the direct fluorescent-antibody assay (DFA) are direct cell-...

Section: Discussionmentioning

confidence: 99%

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Bliem

Schauer

Plicka³

et al. 2015

“…Therefore, characterization of inhibitors by analyzing raw-water sample and final sample concentrates is required for future establishment of a method to improve virus detection efficiency. Rock et al (26) applied EEM analysis to virus extracts obtained from biosolid samples using beef extract and glycine buffer and found peaks of humic acid-like and fulvic acidlike substances. However, the relationship between the virus detection efficiency and the EEM peaks was unclear.…”

Section: Discussionmentioning

confidence: 99%

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

Hata

Katayama

Furumai

2015

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...

“…However, RT-PCR inhibition has been reported to be observed even under these conditions (11,15), probably because of coconcentration of humic acid, which precipitates in the presence of cations (6) or acid conditions and becomes soluble at alkaline pH (23). Moreover, a silica membrane-based nucleic acid extraction/ purification method, which is a commonly used method for molecular detection of viruses in aquatic environments (3,15,41), cannot always effectively remove humic acid from samples (3,34). The principle of this method is based on attachment and detachment of nucleic acids to silica by altering pH and ionic strength (29).…”

mentioning

confidence: 99%

“…Such inhibitory effects also occur in the detection of bacterial genome (7,45). Humic acid, fulvic acid, a humic acid-like component in beef extract, which can be used as an eluent for virus concentration, and cations such as Ca 2ϩ and Fe 3ϩ are known to inhibit RT-PCR (1,15,26,34,49). Microfiltration (MF) membrane-based methods using acid rinse procedures followed by alkaline elution were developed to avoid the use of beef extract (16,21).…”

mentioning

confidence: 99%

Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

Hata

Katayama

Kitajima

et al. 2011

Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primersharing controls.