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Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples.

Rački N, Dreo T, Gutierrez-Aguirre I, Blejec A, Ravnikar M - Plant Methods (2014)

Bottom Line: Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples.This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater.Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia.

ABSTRACT

Background: Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR.

Results: Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR.

Conclusions: This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.

No MeSH data available.


Related in: MedlinePlus

Mean copies per partition (lambda) determined in the control sample (NIC) with no inhibitor added and in the samples spiked with serial dilutions of the inhibitor extracts from the selected matrices and from the chemical inhibitors. H, M, L, (VL), high, medium, low (and very low) concentrations of inhibitors, respectively. Black line denotes the mean value of lambda for NIC.
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Fig3: Mean copies per partition (lambda) determined in the control sample (NIC) with no inhibitor added and in the samples spiked with serial dilutions of the inhibitor extracts from the selected matrices and from the chemical inhibitors. H, M, L, (VL), high, medium, low (and very low) concentrations of inhibitors, respectively. Black line denotes the mean value of lambda for NIC.

Mentions: The mean target copies per partition (droplet) (lambda; [26]) for the RT-ddPCR varied little within replicates for a given inhibitor concentration (average coefficient of variation 7%). However, most of the inhibitors tested led to an underestimation of lambda (Figure 3) in comparison to the no-inhibition control. The seed extract and tannic acid were the inhibitors that most markedly reduced the lambda, compared to the no-inhibition control. This reduction was most likely related to the strong effects exerted by these two inhibitors on the number of positive droplets (Additional file 1: Figure S2), and which resulted in a marked underestimation of the target concentration calculated in these samples (see below, Figures 4A and 5C).Figure 3


Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples.

Rački N, Dreo T, Gutierrez-Aguirre I, Blejec A, Ravnikar M - Plant Methods (2014)

Mean copies per partition (lambda) determined in the control sample (NIC) with no inhibitor added and in the samples spiked with serial dilutions of the inhibitor extracts from the selected matrices and from the chemical inhibitors. H, M, L, (VL), high, medium, low (and very low) concentrations of inhibitors, respectively. Black line denotes the mean value of lambda for NIC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307183&req=5

Fig3: Mean copies per partition (lambda) determined in the control sample (NIC) with no inhibitor added and in the samples spiked with serial dilutions of the inhibitor extracts from the selected matrices and from the chemical inhibitors. H, M, L, (VL), high, medium, low (and very low) concentrations of inhibitors, respectively. Black line denotes the mean value of lambda for NIC.
Mentions: The mean target copies per partition (droplet) (lambda; [26]) for the RT-ddPCR varied little within replicates for a given inhibitor concentration (average coefficient of variation 7%). However, most of the inhibitors tested led to an underestimation of lambda (Figure 3) in comparison to the no-inhibition control. The seed extract and tannic acid were the inhibitors that most markedly reduced the lambda, compared to the no-inhibition control. This reduction was most likely related to the strong effects exerted by these two inhibitors on the number of positive droplets (Additional file 1: Figure S2), and which resulted in a marked underestimation of the target concentration calculated in these samples (see below, Figures 4A and 5C).Figure 3

Bottom Line: Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples.This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater.Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia.

ABSTRACT

Background: Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR.

Results: Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR.

Conclusions: This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.

No MeSH data available.


Related in: MedlinePlus