Chapter Four · failure evidence
What RT-qPCR & Nucleic Acid Amplification got wrong, from 73 dissertations
The collected records demonstrate that nucleic acid amplification and RT-qPCR workflows frequently encounter enzymatic inhibition, non-specific primer binding, and detection limits. They also highlight challenges in validating high-throughput sequencing candidates and the reduced sensitivity of alternative point-of-care assays relative to standard RT-qPCR. These records come from PhD theses at 22 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Non-specific amplification and primer dimers cause false positives in negative controls
Assays frequently suffer from non-target amplification, primer-dimer formation, and elevated background signals in no-template or probe-free controls. These off-target cross-reactions are often exacerbated by non-specific primer interactions, elevated reaction temperatures, high magnesium concentrations, or non-stringent probe designs.
Tried and failed
pH-based detection of isothermal nucleic acid amplification applied to bacterial pathogen detection. Outcome: no signal. Reason: buffering capacity and primer-dimer noise obscured pH changes between target and negative controls
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Tried and failed
dye-based quantitative real-time PCR applied to gene expression in hydrogel-encapsulated fibroblasts. Outcome: no signal. Reason: Severe non-target amplification, high background in no-reverse-transcription controls, and poor replicate consistency.
Developing a hydrogel model to study the role of environmental factors on pulmonary fibroblasts · UT Austin
Considered and rejected
Considered and rejected: Rejected HDA primer pairs for SS recN (recN_F4/R4) due to false positive amplification in no-template controls or extremely poor analytical sensitivity (200,000 copies/µL).
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Tried and failed
RT-LAMP isothermal amplification primer design applied to target RNA detection. Reason: Non-specific primer interactions caused severe background amplification in no-template controls
Nanobiotechnology Enabled Environmental Sensing of Water and Wastewater · Virginia Tech
Tried and failed
helicase-dependent isothermal amplification assay design applied to bacterial pathogen diagnostic targets. Reason: persistent false-positive non-specific amplification in no-template controls across multiple target genes
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Tried and failed
increasing reaction temperature and magnesium ion concentration applied to reverse transcription loop-mediated isothermal amplification. Reason: caused non-specific amplification in no-template negative controls
Nanobiotechnology Enabled Environmental Sensing of Water and Wastewater · Virginia Tech
Tried and failed
positioning mismatch far from competitive primer terminus applied to allele-specific isothermal nucleic acid amplification. Reason: Placing the mismatch over 4 bp away caused internal bulge formation, leading to non-specific amplification.
Tried and failed
branched DNA and non-split hybridization chain reaction applied to single-molecule DNA fluorescence in situ hybridization. Reason: non-specific amplification produced high background signal in negative controls
MAPPING BACTERIA AND MOBILE GENETIC ELEMENTS IN HUMAN MICROBIOMES · Cornell
Tried and failed
using legacy standard primers in multiplex amplicon sequencing applied to fungal mating-type marker genotyping. Reason: low annealing temperatures caused low amplification efficiency and off-target cross-reactivity in multiplexing
Considered and rejected
Considered and rejected: Rejected relying solely on the Loopamp MTBC LAMP assay or endpoint RD PCR for definitive isolate identification due to non-specific amplification and contaminating non-tuberculous mycobacteria/Actinobacteria.
Bovine Tuberculosis in the Wolaita Zone of Ethiopia · Research Repository UCD
Considered and rejected
Considered and rejected: Rejected SYBR Green qPCR assays in favor of TaqMan fluorogenic probes due to indiscriminate amplification of non-target double-stranded DNA and primer dimers.
Developing a hydrogel model to study the role of environmental factors on pulmonary fibroblasts · UT Austin
Considered and rejected
Considered and rejected: Rejected 74 °C RT-LAMP incubation and 5.0 mM MgSO4 due to non-specific amplification and false positives in NTCs.
Nanobiotechnology Enabled Environmental Sensing of Water and Wastewater · Virginia Tech
Considered and rejected
Considered and rejected: Single-round primary RT-PCR was abandoned in favor of a two-step nested PCR design because single PCR yielded primer-dimers and lacked amplification specificity/sensitivity.
Sequenzierung der Antigen-bindenden Region von natürlich vorkommenden alpha-Synuklein-reaktiven Autoantikörpern Sequencing of the antigen-binding region of naturally occurring alpha-synuclein-reactive autoantibodies · open_UMR Marburg DSpace 10.0
Tried and failed
viral metagenomic screening and targeted pcr amplification applied to wild invertebrate tissue samples. Outcome: no signal. Reason: Target virus was absent; previous positive detections were due to non-specific cross-amplification of a related virus.
An investigation of the echinoderm virome: the ecology and pathogensis of sea star densoviruses · Cornell
Tried and failed
single-probe fluorescence in situ hybridization amplification applied to intracellular viral RNA detection. Reason: nonspecific background amplification generated false positive signals even in probe-free negative controls
Accessing the Viral Cargo: Capsid Dynamics and Disassembly · Harvard
Considered and rejected
Considered and rejected: Quantification of AMF via qPCR was rejected in favour of microscopic gridline intersection counting due to non-specific amplifications and primer-dimer artifacts.
Exploiting mycorrhizal selection of beneficial rhizosphere bacteria from the soil microbiome. · Cranfield
Considered and rejected
Considered and rejected: Discontinued the D17Z-1 centromeric RT-qPCR primer probe due to insufficient signal enrichment of reverse-transcribed RNA over background genomic DNA contamination.
Investigating the role of homologous recombination at human centromeres · Oxford
Flawed primer and reaction designs lead to poor efficiency or amplification failure
Inefficient primer binding, excessive amplification bias toward DNA over RNA, and lack of thermal cycling for conformational probe changes compromise amplification reactions. Researchers frequently encounter complete amplification failure, allele dropout, or reduced yield when using suboptimal primer concentrations, degenerate primers, or integrated one-step protocols.
Lost to a baseline
1 of 16 designed pyrosequencing primer sets failed PCR amplification despite repeated protocol optimisations and was excluded from analysis
Molecular mechanisms of ovarian cancer prevention · Imperial
Considered and rejected
Considered and rejected: Lower primer concentration of 0.2 µl in EqHV qPCR master mix was rejected because it failed to yield detectable amplification for positive controls
Insights into equine hepacivirus: assay development and transmission patterns Improving understanding of the impact and transmission of equine hepacivirus A · University of Nottingham Repository
Considered and rejected
Considered and rejected: Rejected using MAP F Realtime primer for further qPCR experiments due to low query sequence coverage (58.3% of caprine strains < 100%) and poor amplification.
Development and evaluation of new methods to optimise the Actiphage RapidTM assay for detection of mycobacteria · University of Nottingham Repository
Considered and rejected
Considered and rejected: Rejected Q-solution in multiplex PCR because preliminary testing showed reduced amplification efficiency.
Dna Recovery from Challenging Forensic Samples Using Alternative Markers and Next Generation Sequencing · DSpace at SHSU
Considered and rejected
Considered and rejected: Rejected DNA reference molecules in RT-qRPA due to a ~30-fold amplification bias over RNA targets
Development of nucleic acid detection methods for object provenance and viral diagnostics · Harvard
Tried and failed
molecular beacon and FRET probe detection applied to isothermal nucleic acid amplification. Outcome: no signal. Reason: Lack of thermal cycling prevents probe stem conformational changes and random primers interfere with hybridization.
Single-Virus/Single-Cell Sequencing with Droplet Microfluidics · Harvard
Considered and rejected
Considered and rejected: Sequencing solely from long-range PCR products was abandoned in favor of sequencing directly from purified lambda clone DNA due to PCR amplification difficulties and ambiguities
Considered and rejected
Considered and rejected: Discarded CloneAmp Hi-Fi PCR in favor of LongAmp Taq for full-genome amplification due to lack of amplification with CloneAmp
The effect of mutations in the envelope protein of Zika virus on cellular tropism · University of Nottingham Repository
Tried and failed
competitive reverse transcription amplification using DNA standard applied to RNA quantitation. Reason: Amplification strongly biased toward DNA over RNA due to reverse transcription efficiency differences.
Development of nucleic acid detection methods for object provenance and viral diagnostics · Harvard
Tried and failed
degenerate primer PCR targeting alternative marker genes applied to detecting photosynthetic gene clusters in metagenomes. Outcome: worse than baseline. Reason: low amplification efficiency and sensitivity loss on low-quality template DNA compared to apcE2 marker
Global distribution and ancient evolution of far-red photosynthesis · Imperial
Tried and failed
one-step direct in-droplet RT-PCR applied to single-cell transcript linkage. Outcome: worse than baseline. Reason: poor reaction efficiency and low yield compared to decoupled reverse transcription and amplification steps
Methods and Materials to Generate Antibody Repertoire Libraries · Harvard
Considered and rejected
Considered and rejected: Rejected degenerate oligonucleotide-primed PCR (DOP-PCR) whole-genome amplification approaches for single-cell sequencing due to poor genomic coverage and allele dropout.
Genomic Instability at Single Cell Resolution · ResearchWorks
Considered and rejected
Considered and rejected: Smart-seq2 without UMIs for high-throughput single-cell experiments (suffered from low throughput of ~few hundred cells per plate, PCR amplification bias, and inefficient template switching for lowly expressed transcripts)
Tried and failed
emulsion PCR targeted gene amplification applied to bacterial housekeeping gene detection. Outcome: no signal. Reason: primers designed for the target gene failed to produce amplification
Considered and rejected
Considered and rejected: Rejected initial touchdown PCR protocol for SNPs BP 987/1118 due to poor amplification success across samples.
Validierung von potenziellen Biomarkern für die neurodegenerative Erkrankung Spinozerebelläre Ataxie Typ 3 (SCA3) und des krankheitsmodulierenden Einflusses intragenischer Polymorphismen im Gen ATXN3 · Publikationssystem UB Tuebingen
Considered and rejected
Considered and rejected: Rejected using the Dengue_2_ET300 qPCR primer set after experimental trials showed improper amplification compared to Dengue_2_Black.
Determination of the Effects of Microplastic Exposure on Mosquito Traits and Pathogen Progression in Two Vectors · Texas Tech
Tried and failed
quantitative real-time PCR amplification of target gene applied to endothelial cell gene expression quantification. Outcome: no signal. Reason: Target gene could not be amplified or assessed due to technical PCR amplification issues
Disrupted iron homeostasis in the vascular endothelium: implications for health and disease · Imperial
Low target abundance and ultra-low biomass fall below analytical detection limits
Target sequences present in low-abundance samples such as oligotrophic soils, nanobiopsies, and biofluids repeatedly fail to cross assay detection thresholds. In these low-copy or low-biomass regimes, quantitative and endpoint amplification responses become non-linear, highly inconsistent, or yield no detectable signal.
Tried and failed
specific primer PCR amplification applied to anaerobic soil enrichment cultures. Outcome: no signal. Reason: target microorganism sequences were absent or below detection limit in incubated alkaline soil slurries
Coupled iron reduction-ammonium oxidation (Feammox) in alkaline soils polluted with nitrogen · HARVEST
Tried and failed
qPCR amplification of target antibiotic resistance genes applied to plant root slurry samples. Outcome: no signal. Reason: The target gene ermB failed to amplify across all root slurry samples.
Antimicrobial resistance in the environment: transport, transfer, and evolution · Iowa State
Tried and failed
16S rRNA amplicon sequencing applied to low-biomass filter media DNA extracts. Outcome: no signal. Reason: samples exhibited very low to no PCR amplification during quality control
Upstream Effects on Microbial Community Selection in Biological Activated Carbon Filters · unevada
Tried and failed
18S qPCR fungal DNA amplification applied to low-biomass volcanic soil samples. Outcome: no signal. Reason: Fungal biomass was below detection limits in the oligotrophic volcanic tephra and basalt environments.
Assessment of Mars Analogue Instruments and Biosignatures in Icelandic Mars Analogue Environments: Implications for Astrobiology · Georgia Tech
Tried and failed
isothermal nucleic acid amplification for quantification applied to low-copy target nucleic acids. Reason: amplification response becomes non-linear and unreliable below 100 copies per reaction
ADVANCING GLOBAL POINT-OF-CARE DIAGNOSIS: INNOVATIONS IN DETECTING INFECTIOUS DISEASES AND CANCER · Cornell
Tried and failed
Nucleic acid sequence-based amplification applied to low-input single-cell RNA extracts. Outcome: no signal. Reason: Isothermal amplification failed to reliably amplify RNA from extremely low-input nanobiopsy samples
Quantifying breast cancer adaption: platforms for studying transcriptional changes in single cells · Imperial
Tried and failed
Polymerase spiral reaction isothermal amplification applied to bacterial pathogen nucleic acid detection. Outcome: no signal. Reason: Assays exhibited extremely poor analytical sensitivity or completely failed to produce detectable amplification
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Tried and failed
standard endpoint PCR amplification applied to low-pathogen-load field swab samples. Outcome: no signal. Reason: lower analytical sensitivity of endpoint PCR compared to quantitative PCR for low-titer pathogen detection
Tried and failed
single-cell nanopipette biopsy for downstream amplification applied to subcellular cytoplasmic mRNA detection. Outcome: no signal. Reason: insufficient RNA copy number extracted in a single sampling event to yield consistent qPCR amplification
Modified nanotweezers for subcellular extraction of cytoplasmic mrna · Imperial
Tried and failed
direct quantitative PCR without pre-amplification applied to low-abundance cell-free and extracellular RNA. Outcome: no signal. Reason: target transcript copy numbers were too low to cross detection thresholds without prior amplification
Evaluation of Cell-Free RNA as Minimally Invasive Biomarkers of Target Engagement · Harvard
Considered and rejected
Considered and rejected: Single-tube full-length RT-PCR without multiplex amplicon pooling was not viable due to low viral copy numbers and complex food matrices, requiring 4-pool multiplex amplification.
Systematic Surveillance of Hepatitis E Virus in Pig Livers and Risk Assessment Related to Consumption in Selected Provinces of China · Research Repository UCD
Tried and failed
end-point PCR for target gene detection applied to environmental bacterial isolates. Outcome: no signal. Reason: Amplification failed across all suspected isolates and positive control runs.
Dispersión de patógenos ESKAPE en el aire y su detección en efluentes cercanos a hospitales de la ciudad de Puebla · Repositorio Institucional BUAP
Tried and failed
RT-qPCR validation of sequencing biomarker candidates applied to low-abundance biofluid microRNA biomarkers. Outcome: no signal. Reason: Target microRNAs were undetectable in more than half of the biofluid samples
MicroRNAs in extracellular vesicles as biomarkers for pancreaticobiliary cancers · Imperial
Tried and failed
RT-qPCR quantification of circulating target molecules applied to blood-based biomarker discovery. Outcome: no signal. Reason: Target molecules had undetectable or very low expression in over 50% of samples.
Expression levels of blood microRNAs as biomarker of cognitive decline due to Alzheimer's disease · Imperial
Novel isothermal and point-of-care platforms lose sensitivity or speed compared to RT-qPCR
Alternative amplification strategies including LAMP, RPA, and CRISPR-based assays frequently exhibit higher limits of detection and false-negative outcomes on low-titer samples when compared directly against standard RT-qPCR. Furthermore, on-chip adaptations and modified primers often suffer from delayed amplification times and slower reaction kinetics than conventional thermocyclers.
Lost to a baseline
Duplex SHINE H275Y/H275 assay exhibited false-negative results on two patient samples with high Ct (>35) values compared to RT-qPCR
Lost to a baseline
At 62°C on hot plate, dLAMP amplification time in microwells (21 min) was not lower than conventional LAMP in a commercial thermocycler
Production of polymer surfaces for in vitro diagnostics application by micro-injection moulding · Research Repository UCD
Lost to a baseline
Lyophilised LAMP displayed a slightly delayed amplification (higher time-to-positive) compared to freshly prepared wet reactions.
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Lost to a baseline
P5-P6, P8-P9, and P13-P14 doubly dye-labelled LAMP primers showed slower amplification kinetics than unmodified control primers (P1).
Synthesis and studies on modified oligonucleotides for diagnostic and therapeutic applications · Oxford
Lost to a baseline
dLAMP CMV amplification on-chip at low heating temperatures took almost an hour, more than double the reaction time of conventional LAMP in a thermocycler
Production of polymer surfaces for in vitro diagnostics application by micro-injection moulding · Research Repository UCD
Lost to a baseline
RT-qLAMP achieved 91% sensitivity for SARS-CoV-2 detection, beaten by RT-qPCR (12 false negatives among 183 clinical samples in low concentration brackets).
New molecular methods for point-of-care infectious disease diagnosis · Imperial
Lost to a baseline
LwaCas13a CrisprZyme without pre-amplification yielded a higher LOD (7.88 pM) compared to rtRPA-CrisprZyme (8.30 fM) or LbuCas13a-CrisprZyme (264 fM)
Amplification strategies for CRISPR-based diagnostics · Imperial
Tried and failed
multiplex RT-qPCR diagnostic kit evaluation applied to viral RNA detection across Ct ranges. Outcome: worse than baseline. Reason: lower sensitivity and performance across the full cycle threshold range compared to alternative commercial assays
Comparative Assessment of SARS-CoV-2 Diagnostics in Nigeria · Harvard
Lost to a baseline
STOPCovid.v1 lateral flow sensitivity was lower than RT-qPCR, capturing only 60.5% of RT-qPCR-positive patients (false negatives at Ct > 32).
Characterization and Engineering of Transposons for Genome Editing · MIT
Lost to a baseline
STOPCovid.v2 reaction chemistry itself is '20 times less sensitive than the RT-qPCR reaction itself' (100 copies vs 5 copies per reaction).
Characterization and Engineering of Transposons for Genome Editing · MIT
Lost to a baseline
RT-qLAMP achieved 90.55% sensitivity for SARS-CoV-2 detection, beaten by RT-qPCR's 100% sensitivity (12 of 127 RT-qPCR positive samples were missed by RT-qLAMP in low viral load brackets)
New molecular methods for point-of-care infectious disease diagnosis · Imperial
Lost to a baseline
LAMPcov achieved 90.55% sensitivity, missing 12 low-viral-load clinical samples detected by CDC N1 RT-qPCR
Matrix inhibitors and lysis buffer carryover suppress enzymatic amplification
Amplification enzymes in PCR, RT-qPCR, and isothermal assays are repeatedly inhibited by components present in environmental matrices, bodily fluids, and unpurified cell extracts. Carryover of extraction chemicals such as high concentrations of tRNA, DTT, EGTA, salts, and detergents directly diminishes or abolishes fluorescent signal and target amplification.
Tried and failed
nanoliter microfluidic high-throughput quantitative PCR applied to inhibitor-rich environmental manure samples. Outcome: no signal. Reason: Extreme nanoliter reaction volumes heightened susceptibility to sample PCR inhibitors, preventing target amplification.
Characterization of antimicrobial resistance in agricultural landscapes · Iowa State
Tried and failed
standard environmental master mix PCR amplification applied to marine environmental DNA samples. Outcome: no signal. Reason: natural PCR inhibitors in coastal seawater samples prevented successful target amplification
Diversity of fishes along the Texas Gulf Coast using environmental DNA metabarcoding · Texas Tech
Tried and failed
enzymatic amplification in polyacrylic acid hydrogels applied to in situ transcriptomics. Outcome: no signal. Reason: negatively charged polyacrylic acid networks inherently inhibit downstream reverse transcription and rolling circle amplification enzymes
Spatially precise in situ transcriptomics in intact biological systems · MIT
Tried and failed
adding salts and detergents to reaction buffer applied to RT-qPCR amplification. Outcome: worse than baseline. Reason: salts and detergents reduced RT-qPCR fluorescence signal
Ribosomal Structure, Function, and Trafficking · Georgia Tech
Considered and rejected
Considered and rejected: Rejected relying on raw undiluted field DNA extracts due to total PCR inhibition; required 1:10 dilution to achieve amplification.
Advancing eDNA methods for monitoring sharks and rays · Imperial
Considered and rejected
Considered and rejected: Rejected direct RT-qPCR without extraction due to reduced sensitivity from inhibitors in bodily fluids.
Delamination-based Control of Capillary Flow for Physically Programmed Lateral Flow Assays · Georgia Tech
Considered and rejected
Considered and rejected: Rejected using high concentrations of tRNA (>6 µg) in lysis buffer because excess carried-over tRNA inhibited downstream RT-qPCR amplification.
Development of platforms for detection of single nucleotide polymorphisms: applications on pathogens, genetic diseases, and beyond · ResearchWorks
Tried and failed
lysis with high concentration DTT and EGTA applied to downstream isothermal nucleic acid amplification. Outcome: no signal. Reason: the high DTT and EGTA concentrations inhibited downstream loop-mediated isothermal amplification (LAMP) reactions
CRISPR Biosensors for Resource-limited Nucleic Acid Detection · MIT
Tried and failed
increasing unpurified crude lysate input volume fraction applied to single-tube isothermal nucleic acid amplification detection. Outcome: worse than baseline. Reason: carryover lysis buffer components inhibited the downstream enzymatic amplification and detection reaction
Lost to a baseline
Direct qPCR via heat treatment (Protocols 1-3) yielded severely lower sensitivity and higher Cts than standard extraction/amplification (Protocol 4; e.g., 0/8 vs 8/8 detected for PRRSV neat).
Assessment of strategies to improve routine PCR diagnostics for swine pathogens · Iowa State
Tried and failed
colony PCR directly on whole bacterial cells applied to plasmid verification in Gram-negative bacteria. Outcome: no signal. Reason: intact cells or cell lysis components inhibited amplification, requiring purified plasmid DNA instead
Recombineering for Precise Genome Editing in Shewanella oneidensis · Cornell
Measurement variability and cDNA noise prevent validation of high-throughput sequencing hits
Targeted RT-qPCR assays often fail to replicate differential expression hits from bulk RNA sequencing due to high measurement noise, media batch variability, and small sample cohorts. Standard curves and random-primed cDNA synthesis protocols also suffer from assay-to-assay variability and lack of linearity at high dilutions.
Tried and failed
RT-qPCR validation of RNA-seq differential expression applied to candidate gene expression validation. Outcome: no signal. Reason: bulk RNA-seq differential expression hit failed to replicate upon targeted RT-qPCR quantification
Tried and failed
whole-transcriptome random-primed cDNA synthesis for RT-qPCR applied to bacterial inducible gene cluster quantification. Outcome: unstable. Reason: standard cDNA kits yielded inconsistent amplification requiring gene-specific priming
Identification and characterization of unique enzymatic transformations by gut microbes · Harvard
Tried and failed
RT-qPCR standard curve quantification in pure water applied to low-concentration viral RNA detection. Outcome: unstable. Reason: Significant assay-to-assay variability in slopes and intercepts with loss of linearity at high dilutions
Wastewater Surveillance for Emerging Infectious Diseases: Optimization and Implementation of Methods · ResearchWorks
Tried and failed
RT-qPCR validation of siRNA knockdown applied to measuring targeted gene silencing efficiency. Outcome: worse than baseline. Reason: exhibited high sample variability and lower robustness compared to western blotting
Tried and failed
RT-qPCR validation of RNA-seq differential expression applied to differentially expressed genes in clinical samples. Outcome: no signal. Reason: High inter-subject variability combined with small sample size prevented validation results from reaching statistical significance
Tried and failed
RT-qPCR quantification of viral RNA targets applied to wastewater solids for epidemiological correlation. Outcome: no signal. Reason: lack of statistically significant correlation between target gene concentrations and reported clinical case numbers
Monitoring SARS-CoV-2 in Municipal Wastewater and Correlation of Results with Covid-19 Cases from Five Municipalities in Ontario, Canada · Carleton University Institutional Repository
Tried and failed
RT-qPCR validation of RNA-Seq differential expression candidates applied to candidate biomarker genes. Outcome: no signal. Reason: candidate genes failed to reach statistically significant differential expression after multiple testing correction
Discovery and validation of host transcriptomic and proteomic biomarkers for paediatric tuberculosis · Imperial
Tried and failed
in-house RT-qPCR assay on legacy instrumentation applied to viral RNA quantification. Reason: Reagent contamination, temperature excursions during transport, and platform incompatibility produced uninterpretable results
Assessing and improving the Hepatitis C virus cascade of care for marginalised populations · Imperial
Considered and rejected
Considered and rejected: Abandoned RT-qPCR gene expression assessment in favour of NanoString due to high measurement variability, lack of internal calibrators, and cDNA-step noise.
Left open by the authors
Problems the authors named and did not get to.
Left open
Perform targeted amplification of high-abundance species to track their response to experimental treatments and evaluate founder effects. Blocker: Requires a wet lab and physical soil/manure samples to perform targeted PCR amplification.
Impact of manure amendment on species replacement patterns in soil microbial communities · Iowa State
Left open
Compare the amplification efficiency of PM_B assay with and without loop primers. Blocker: Requires a wet lab and biological reagents/samples to perform isothermal amplification assays.
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Left open
Evaluate degenerate primer bases instead of triplex isothermal amplification assays for the detection of divergent Streptococcus suis strains. Blocker: Requires wet-lab experimental validation and pathogen DNA samples for isothermal amplification assays.
The development of a point-of-care diagnostic for the porcine respiratory disease complex · Imperial
Left open
Design tiled isothermal multiplexed RPA primers and protocols to replace PCR thermocycling for point-of-care WATSON diagnostics. Blocker: Requires wet lab experimentation and physical validation of isothermal amplification reagents and assays
Left open
Optimize isothermal amplification routes to amplify ultra-low input RNA prior to single-cell RNA sequencing. Blocker: Requires wet lab equipment, biological samples, and sequencing reagents to perform RNA amplification experiments
Quantifying breast cancer adaption: platforms for studying transcriptional changes in single cells · Imperial
Left open
Develop methods for one-pot nucleic acid amplification and detection coexisting within aqueous two-phase system protocell reactions. Blocker: Requires a wet lab, molecular biology reagents (CFE, nucleic acid amplification enzymes), and experimental microfluidics/microwell apparatus.
New Interfaces To Advance Point-Of-Care Biosensor Diagnostics · Georgia Tech
Left open
Develop high-throughput RT-qPCR assays to screen LNA probe length and disruption effects on G-quadruplex-induced DNA polymerase stalling. Blocker: Requires a molecular biology wet laboratory, RT-qPCR instrumentation, and chemical synthesis of LNA oligonucleotides.
Left open
Validate isothermal miRNA detection methods on endogenous clinical miRNA directly from real biological samples. Blocker: Requires clinical biological samples and a wet lab equipped for isothermal nucleic acid amplification
Left open
Evaluate the multi-feature nucleic acid quantification framework on experimental isothermal amplification curves. Blocker: Requires wet-lab experimental data or generation of isothermal amplification curves.
Intelligent algorithms for DNA detection, quantification and multiplexing · Imperial
Left open
Evaluate the diagnostic performance of the Allplex SARS-CoV-2 RT-qPCR kit specifically at low Ct levels. Blocker: Requires physical clinical samples, laboratory diagnostics infrastructure, and RT-qPCR kits
Comparative Assessment of SARS-CoV-2 Diagnostics in Nigeria · Harvard
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