PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions

ddPCR is getting more and more popular in the field of eDNA-based aquatic monitoring. Even if emulsion PCR used in ddPCR confers a partial resistance to inhibition due to the high number of reactions for the same sample (between 10K and 20K), it is not impervious to it. Inhibition impacts the fluorescence amplitude of positive droplets, affecting both their dispersion and their position relatively to the negative droplets cloud. This fluctuation could jeopardize the use of a shared threshold among several samples and thus the objective assignation of the positive droplets. This is even more critical for low concentration samples such as eDNA samples: the positive droplets are scarce and it is thus crucial to objectively discriminate if they can be counted as positive by establishing an appropriate threshold. Another issue is the artifactual generation of high fluorescence droplets that could be counted as positive with a single threshold solution. Here we propose a double threshold method to take both high fluorescence droplets and PCR inhibition impact into account allowing for an objective sorting of the positive and negative droplets in ddPCR assays.

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Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

10.1101/2021.10.19.464972 ◽

2021 ◽

Author(s):

Shunsuke Matsuoka ◽

Yoriko Sugiyama ◽

Mariko Nagano ◽

Hideyuki Doi

Keyword(s):

Dna Extraction ◽

Environmental Dna ◽

Operational Taxonomic Unit ◽

Pcr Inhibition ◽

Dna Metabarcoding ◽

Fungal Dna ◽

Diversity Studies ◽

Microbial Dna ◽

Methodological Studies ◽

Biodiversity Analysis

Background: Environmental DNA (eDNA) metabarcoding is a rapidly expanding technique for efficient biodiversity monitoring, especially of animals. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of the DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results [i.e., operational taxonomic unit (OTU) number and composition] of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

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Substances Affecting PCR: Inhibition or Enhancement

PCR: Clinical Diagnostics and Research ◽

10.1007/978-3-642-77492-8_4 ◽

1992 ◽

pp. 51-60 ◽

Cited By ~ 4

Author(s):

Arndt Rolfs ◽

Irmela Schuller ◽

Ulrich Finckh ◽

Ines Weber-Rolfs

Keyword(s):

Pcr Inhibition

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Polymerase Chain Reaction Assay With Urine Specimens in the Diagnosis of AcuteChlamydia trachomatisInfection in Women

Infectious Diseases in Obstetrics and Gynecology ◽

10.1155/s1064744996000531 ◽

1996 ◽

Vol 4 (5) ◽

pp. 276-280

Author(s):

R. Pasternack ◽

A. Mustila ◽

P. Vuorinen ◽

P. K. Heinonen ◽

A. Miettinen

Keyword(s):

Polymerase Chain Reaction ◽

Pcr Inhibition ◽

Chain Reaction ◽

Pcr Assay ◽

Cervical Swab ◽

Female Urine ◽

Increased Sensitivity ◽

Full Benefit ◽

Polymerase Chain ◽

Urine Specimens

Objective:The purpose of this study was to evaluate the benefits achievable by Amplicor polymerase chain reaction (PCR) (F. Hoffmann-LaRoche Ltd., Basel, Switzerland) with urine specimens in addition to PACE 2 (Gen-Probe, Inc., San Diego, California) assay with cervical swab specimens in the diagnosis ofChlamydia trachomatisin women.Methods:Cervical and urine specimens from 286 women were tested forC. trachomatisby PACE 2 and Amplicor PCR, respectively. All urine specimens were analyzed undiluted and diluted 1:10 to detect and eliminate possible PCR inhibition. A confirmatory PCR assay using major outer membrane protein-based primers (MOMP-PCR) was used on urine specimens that were positive by PCR from women who were negative by PACE 2 with cervical swab specimens.Results:Of the endocervical specimens, 26 were positive by the PACE 2 assay. The PCR with urine was positive in 21 of these patients. When the urine specimens were analyzed diluted 1:10, 4 of the 5 PCR-negative specimens from PACE 2-positive patients turned positive by the PCR. Additionally, 4 urine specimens from PACE 2-negative women were positive by the PCR with urine, and 3 of them could be confirmed by MOMP-PCR. Altogether, 29 women were found to be positive forC. trachomatisby either of the two assays.Conclusions:By using the PCR with urine specimens, an 11% increase in sensitivity could be achieved in addition to that obtained by PACE 2 assay with cervical swab specimens. In the present material, however, the increased sensitivity was reversed by the presence of PCR inhibitors in 14% of the female urine specimens. Amplicor PCR with urine specimens can undoubtedly be recommended for the diagnosis of chlamydial infections in women. However, constant monitoring of the PCR inhibition seems highly advisable to obtain full benefit of the sensitivity of the PCR.

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