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Missing the Match Might Not Cost You the Game: Primer-Template Mismatches Studied in Different Hepatitis A Virus Variants.

ABSTRACT: Mismatches between template sequences and reverse transcription (RT) or polymerase chain reaction (PCR) primers can lead to underestimation or false negative results during detection and quantification of sequence-diverse viruses. We performed an in silico inclusivity analysis of a widely used RT-PCR assay for detection of hepatitis A virus (HAV) in food, described in ISO 15216-1. One of the most common mismatches found was a single G (primer) to U (template) mismatch located at the terminal 3'-end of the reverse primer region. This mismatch was present in all genotype III sequences available in GenBank. Partial HAV genomes with common or potentially severe mismatches were produced by in vitro transcription and analysed using RT-ddPCR and RT-qPCR. When using standard conditions for RT-qPCR, the mismatch identified resulted in underestimation of the template concentration by a factor of 1.7-1.8 and an increase in 95% limit of detection from 8.6 to 19 copies/reaction. The effect of this mismatch was verified using full-length viral genomes. Here, the same mismatch resulted in underestimation of the template concentration by a factor of 2.8. For the partial genomes, the presence of additional mismatches resulted in underestimation of the template concentration by up to a factor of 232. Quantification by RT-ddPCR and RT-qPCR was equally affected during analysis of RNA templates with mismatches within the reverse primer region. However, on analysing DNA templates with the same mismatches, we found that ddPCR quantification was less affected by mismatches than qPCR due to the end-point detection technique.

SUBMITTER: Persson S

PROVIDER: S-EPMC6689102 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Project description:The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3-12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled "Polymerase-exonuclease (PEX) PCR", in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

Project description:Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Project description:BackgroundVisual search declines with aging, dementia, and brain injury and is linked to limitations in everyday activities. Recent studies suggest that visual search can be improved with practice using computerized visual search tasks and puzzle video games. For practical use, it is important that visual search ability can be assessed and practiced in a controlled and adaptive way. However, commercial puzzle video games make it hard to control task difficulty, and there are little means to collect performance data.ObjectiveThe aim of this study was to develop and initially validate the search and match task (SMT) that combines an enjoyable tile-matching match-3 puzzle video game with features of the visual search paradigm (taskified game). The SMT was designed as a single-target visual search task that allows control over task difficulty variables and collection of performance data.MethodsThe SMT is played on a grid-based (width × height) puzzle board, filled with different types of colored polygons. A wide range of difficulty levels was generated by combinations of 3 task variables over a range from 4 to 8 including height and width of the puzzle board (set size) and the numbers of tile types (distractor heterogeneity). For each difficulty level, large numbers of playable trials were pregenerated using Python. Each trial consists of 4 consecutive puzzle boards, where the goal of the task is to find a target tile configuration (search) on the puzzle board and swap 2 adjacent tiles to create a line of 3 identical tiles (match). For each puzzle board, there is exactly 1 possible match (single target search). In a user study with 28 young adults (aged 18 to 31 years), 13 older (aged 64 to 79 years) and 11 oldest (aged 86 to 98 years) adults played the long (young and older adults) or short version (oldest adults) of the difficulty levels of the SMT. Participants rated their perception and the usability of the task and completed neuropsychological tests that measure cognitive domains engaged by the puzzle game.ResultsResults from the user study indicate that the target search time is associated with set size, distractor heterogeneity, and age. Results further indicate that search performance is associated with general cognitive ability, selective and divided attention, visual search, and visuospatial and pattern recognition ability.ConclusionsOverall, this study shows that an everyday puzzle game-based task can be experimentally controlled, is enjoyable and user-friendly, and permits data collection to assess visual search and cognitive abilities. Further research is needed to evaluate the potential of the SMT game to assess and practice visual search ability in an enjoyable and adaptive way. A PsychoPy version of the SMT is freely available for researchers.

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Missing the Match Might Not Cost You the Game: Primer-Template Mismatches Studied in Different Hepatitis A Virus Variants.

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