Primer Mismatches Research Articles

Evaluation of Loopamp Leishmania detection kit for the diagnosis of cutaneous leishmaniasis in Ethiopia

BackgroundCutaneous leishmaniasis (CL) in Ethiopia and some parts of Kenya is predominantly caused by Leishmania aethiopica. While skin-slit (SS) microscopy is routinely used for CL diagnosis, more sensitive molecular tests are available. The Loopamp™ Leishmania detection kit (Loopamp) is a robust loop-mediated isothermal amplification (LAMP) assay with the potential for implementation in primary healthcare facilities. In this study, we comparatively assessed the diagnostic accuracy of four methods currently used to diagnose CL: Loopamp, kinetoplast DNA (kDNA) PCR, spliced leader RNA (SL-RNA) PCR and SS microscopy.MethodsA study on 122 stored tape disc samples of suspected CL patients was conducted in Gondar, northwestern Ethiopia. Routine SS microscopy results were obtained from all patients. Total nucleic acids were extracted from the tapes and subjected to PCR testing targeting kDNA and SL-RNA, and Loopamp. Diagnostic accuracy was calculated with SS microscopy as a reference test. The limit of detection (LoD) of Loopamp and kDNA PCR were determined for cultured L. aethiopica and Leishmania donovani.ResultsOf the 122 patients, 64 (52.5%) were identified as CL cases based on SS microscopy. Although the PCR tests showed a sensitivity of 95.3% (95% confidence interval [CI] 91.6–99.1), Loopamp only had 48.4% (95% CI 39.6–57.3) sensitivity and 87.9% (95% CI 82.1–93.7) specificity. The LoD of Loopamp for L. donovani was 100-fold lower (20 fg/µl) than that for L. aethiopica (2 pg/µl).ConclusionsThe Loopamp™ Leishmania detection kit is not suitable for the diagnosis of CL in Ethiopia, presumably due to a primer mismatch with the L. aethiopica 18S rRNA target. Further research is needed to develop a simple and sensitive point-of-care test that allows the decentralization of CL diagnosis in Ethiopia.Graphical

Biochemical simulation of mutation synthesis and repair during SARS-CoV-2 RNA polymerization

We biochemically simulated the mutation synthesis process of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex (nsp7/nsp8/nsp12) involving two sequential mechanistic steps that occur during genomic replication: misinsertion (incorporation of incorrect nucleotides) and mismatch extension. Then, we also simulated mismatch repair process catalyzed by the viral nsp10/nsp14 ExoN complex. In these mechanistic simulations, while SARS-CoV-2 RdRp displays efficient mutation synthesis capability, the viral ExoN complex was able to effectively repair the mismatch primers generated during the mutation synthesis. Also, we observed that the delayed RNA synthesis induced by mutation synthesis process was rescued by the viral ExoN activity. Collectively, our biochemical simulations suggest that SARS-CoV-2 ExoN complex may contribute to both maintenance of proper viral genetic diversity levels and successful completion of the viral large RNA genome replication by removing mismatches generated by the viral RdRp.

Characterization, Molecular Mechanism of Prochloraz-Resistance in Fusarium fujikuroi and Development of Loop-Mediated Isothermal Amplification Rapid Detection Technique Based on the S312T Genotype of Resistances.

Rice bakanae disease (RBD) is a typical seed-borne fungal disease caused by Fusarium fujikuroi. Prochloraz is a sterol demethylation inhibitor, which is among the most important classes of active ingredients for the management of RBD. In 2022, the total resistance frequency of F. fujikuroi to prochloraz in Zhejiang Province was 62.67%. The fitness of the prochloraz-resistant population was lower than that of the susceptible population, but its pathogenicity was slightly stronger. The S312T and F511S double mutations of Ffcyp51b were detected in the resistant isolates. Loop-mediated isothermal amplification (LAMP) technology based on S312T was established to rapidly determine prochloraz resistance in F. fujikuroi. LAMP primer mismatch design was performed based on the cyp51b gene, and 100-300 bp sequences containing a mutation at codon 312 were amplified. In a 25 µL reaction tube, 1 pg/µL DNA of F. fujikuroi could be detected. The detection limit for the frequency of prochloraz resistance was 0.498% using this method. We performed LAMP detection on rice seedlings inoculated with prochloraz-sensitive and -resistant isolates and treated them with prochloraz. Prochloraz demonstrated good control in rice seedlings. A chromogenic reaction was observed in seedlings treated with prochloraz-resistant isolates, and the results were verified using electrophoresis. It has been demonstrated that LAMP technology based on the S312T genotype can quickly and specifically detect prochloraz-resistant isolates in rice seedlings.

Twenty-three years of PCR-based seafood authentication assay development: What have we learned?

Seafood is a prime target for fraudulent activities due to the complexity of its supply chain, high demand, and difficult discrimination among species once morphological characteristics are removed. Instances of seafood fraud are expected to increase due to growing demand. This manuscript reviews the application of DNA-based methods for commercial fish authentication and identification from 2000 to 2023. It explores (1) the most common types of commercial fish used in assay development, (2) the type of method used, (3) the gene region most often targeted, (4) provides a case study of currently published assays or primer-probe pairs used for DNA amplification, for specificity, and (5) makes recommendations for ensuring standardized assay-based reporting for future studies. A total of 313 original assays for the detection and authentication of commercial fish species from 191 primary articles published over the last 23 years were examined. The most explored DNA-based method was real-time polymerase chain reaction (qPCR), followed by DNA sequencing. The most targeted gene regions were cytb (cytochrome b) and COI (cytochrome c oxidase 1). Tuna was the most targeted commercial fish species. A case study of published tuna assays (n=19) targeting the cytb region found that most assays were not species-specific through in silico testing. This was conducted by examining the primer mismatch for each assay using multiple sequence alignment. Therefore, there is need for more standardized DNA-based assay reporting in the literature to ensure specificity, reproducibility, and reliability of results. Factors, such as cost, sensitivity, quality of the DNA, and species, should be considered when designing assays.

New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales)

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata

Persicaria odorata is an herbaceous plant with antifungal and antibacterial properties. The plant produces secondary metabolites, including phenols, sulphur-containing compounds and terpenoids, in response to biotic and abiotic stresses. Terpenoids in P. odorata are synthesized through the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. 1-deoxy-D-xylulose-5phosphate isomerase (DXR) is a rate-limiting enzyme in the MEP pathway and may be regulated by microRNA (miRNA) miR398. There is a lack of evidence showing miR398 regulation in terpenoid biosynthesis in P. odorata through DXR-targeting. The study aimed to verify the stem-loop structure of miR398 and analyse its expression towards the target gene, DXR, in treated and control P. odorata. RNA was quantified and qualitatively analysed using a Nanodrop spectrophotometer and agarose gel electrophoresis. The stem loop of miR398 was verified using reverse transcriptase PCR (RT-PCR) and agarose gel electrophoresis. The expression of miR398 and DXR was compared between control and treated samples. Treated leaves were punctured with needles and left for 48h before harvest. Gene expressions were quantified and normalised using reference genes. The stem-loop structure of miR398 was confirmed, despite possible primer mismatches and unspecific binding. This step was essential before comparing and assessing the gene expression of miR398 and DXR. A decrease in abundance of miR398 whereas in increase in abundance was in the treated sample compared to the control sample indicating that miR398 negatively regulated DXR under stress conditions, suggesting an increase in terpenoid synthesis as a defence mechanism. DXR acts as a rate-limiting enzyme in the MEP metabolic pathway. Further studies are needed to quantify the effects of miR398 on terpenoid biosynthesis after wounding in P. odorata.

Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

452. Ongoing Surveillance of SARS-CoV-2 for In Silico Analysis to Monitor the Performance of SARS-CoV-2 Assays in BIOFIRE® Panels

Abstract Background In response to the COVID-19 pandemic, bioMérieux has incorporated assays to detect SARS-CoV-2 into the BIOFIRE® Respiratory 2.1 (RP2.1) Panel, the BIOFIRE® SPOTFIRE® Respiratory (R) Panel and the BIOFIRE® SPOTFIRE® Respiratory (R) Panel Mini. All panels use two assays for SARS-CoV-2 detection, each targeting a different gene. Positive detection in only one of the two assays is required for a positive SARS-CoV-2 result. As new variants of SARS-CoV-2 emerge, in silico analysis remains crucial to ensure that both SARS-CoV-2 assays are reactive to circulating strains. Methods Sequence data (https://gisaid.org/) for SARS-CoV-2 variants deemed significant by the WHO and UKHSA are analyzed monthly. Assay primer regions are assessed for mismatches using Geneious Prime® as well as proprietary software tools. When primer mismatches with potential to affect the sensitivity of SARS-CoV-2 detection are identified (mismatches occurring in the 3’ half of a primer in both assays), and meet the testing criteria, the BIOFIRE RP2.1 Panel is used to empirically evaluate the impact by comparing synthetic templates with mismatches of concern to synthetic template without mismatches. Results As of March 21, 2023, nearly 1,000 variants and 12,744,136 sequences have been analyzed. Of these, only 481 sequences (0.0038%) contain paired mismatches of concern. Wet testing showed < 10x lower projected sensitivity for 288 (72 unique) sequences, 10-100x lower sensitivity for 16 (10 unique) sequences, and only 1 sequence had 100-1000x lower projected sensitivity. Lineage defining mutations were seen within a SARS-CoV-2 assay primer region in 27 variants. However, most lack mutations in the second assay, indicating no major risk for SARS-CoV-2 detection. Wet testing shows that the inclusion of two SARS-CoV-2 assays in the panels helps to mitigate the effect of mismatches of concern. Conclusion Based on the comprehensive in silico analysis of available sequences and variants, BIOFIRE RP2.1 Panel, SPOTFIRE R Panel and SPOTFIRE R Panel Mini continue to function as intended with >99.99% detection of SARS-CoV-2 sequences. Disclosures Eleanor K. Horrocks, bioMerieux: employee|bioMerieux: Stocks/Bonds Toma Todorov, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Alexandra Debernardi, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Usha Spaulding, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Tanner Robinson, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Jeremiah Antosch, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Zhenmei Lu, n/a, bioMerieux: employee|bioMerieux: Stocks/Bonds Matthew Jones, MS, bioMerieux: employee|bioMerieux: Stocks/Bonds Joann Cloud, PhD, bioMerieux: employee|bioMerieux: Stocks/Bonds

Sequence optimized diagnostic assay for Ebola virus detection

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Genotyping of Macaque Population on the Brain-Derived Neurotrophic Factor Gene Polymorphism by Mismatch Amplification Mutation Assay (MAMA)-PCR.

Human rs6265 (196G>A) polymorphism in the BDNF gene is associated with many clinically significant phenotypic manifestations. Rhesus monkey (Macaca mulatta) has a functionally significant rs309950446 ( 136G>A) polymorphism. To determine this polymorphism in macaques, we used mismatch amplification mutation assay (MAMA)-PCR method with non-complementary nucleotide to the template chain at the 3rd position from the 3'-end of the allele-specific primers (mismatch primers), which allowed the best discrimination of the alleles. Genotyping of male rhesus monkeys (n=178) and cynomolgus monkeys (Macaca fascicularis) (n=90) was carried out. The A/A, G/G, and G/A genotypes were found in 16, 34, and 50% rhesus macaques, respectively. In the cynomolgus macaques, the mutant polymorphic allele was not detected. The study results allow considering rhesus macaques as a potential biological model for assessment of the gen-environment interaction of the BDNF gene polymorphism.

Fast genotyping of K-RAS codons 12 and 13 based on streptavidin magnetic microbeads equipped with biotin-coupled single base-pair mismatch PCR (SM-PCR)

In this study, a single base-pair mismatch polymerase chain reaction (SM-PCR) combined with streptavidin magnetic microbeads (MB) for genotyping of codons 12 and 13 of K-RAS gene was established. It was performed by adding three primers including codon 12 forward single-base mismatch primer, codon 13 forward single-base mismatch primer and one universal reverse primer to differentiate single nucleotide polymorphisms. The both forward primers were designed to carry a single-base mismatch nucleotide to either codons 12 and 13. This design was included to increase the primer specificity and to enhance the discrimination power of the point mutation. Two restriction enzymes (StyD4I and HaeIII) were available for double digestion of codons 12 and 13 at the single tube. The wild type of codon 12 and 13 was digested, but the mutant type was not. And then, using the magnetic microbeads for purifying. Finally, wild type of codon 12 and 13 have different emission wavelength, but mutant type did not due to no digestion. This method showed a good consistency between genotyping results and direct sequencing results. The SM-PCR combined with MB method was feasible and efficient for screening of K-RAS codons 12 and 13 in populations.

A novel metric to improve mismatched primer selection and quantification accuracy in amplifying DNA repeats for quantitative polymerase chain reactions.

In quantitative polymerase chain reaction (qPCR) experiments, primers containing mismatches with respect to the template are widely used in measuring repetitive DNA elements. Primer-template mismatches may lead to underestimation of the input sample quantity due to inefficient annealing and amplification. But how primer-template mismatches affect quantification accuracy has not been rigorously investigated. In this study, we performed a series of qPCR experiments in which we tested three pairs of mismatched telomere primers (tel1/tel2, tel1b/tel2b and telg/telc) and two pairs of perfect-match reference gene primers (36B4-F/-R and IFNB1-F/-R) at three different primer concentrations under four cycling conditions. Templates used were genomic DNA from two human cell lines and oligo duplexes which contained telomere sequences, reference gene sequences, or both. We demonstrated that the underestimation of input sample quantity from reactions containing mismatched primers was not due to lower amplification efficiency (E), but due to ineffective usage of the input sample. We defined a novel concept of amplification efficacy (f) which quantifies the effectiveness of input sample amplification by primers. We have modified the conventional qPCR kinetic formula to include f, which corrects the effects of primer mismatches. We demonstrated that reactions containing mismatched telomere primer pairs had similar efficiency (E), but varying degrees of reduced efficacy (f) in comparison to those with the perfect-match gene primer pairs. Using the quantitative parameter f, underestimation of initial target by telomere primers can be adjusted to provide a more accurate measurement. Additionally, we found that the tel1b/tel2b primer set at concentration of 500 nM and 900 nM exhibited the best amplification efficacy f. This study provides a novel way to incorporate an evaluation of amplification efficacy into qPCR analysis. In turn, it improves mismatched primer selection and quantification accuracy in amplifying DNA repeats using qPCR methods.

The global depth range of marine fishes and their genetic coverage for environmental DNA metabarcoding.

The bathymetric and geographical distribution of marine species represent a key information in biodiversity conservation. Yet, deep-sea ecosystems are among the least explored on Earth and are increasingly impacted by human activities. Environmental DNA (eDNA) metabarcoding has emerged as a promising method to study fish biodiversity but applications to the deep-sea are still scarce. A major limitation in the application of eDNA metabarcoding is the incompleteness of species sequences available in public genetic databases which reduces the extent of detected species. This incompleteness by depth is still unknown. Here, we built the global bathymetric and geographical distribution of 10,826 actinopterygian and 960 chondrichthyan fish species. We assessed their genetic coverage by depth and by ocean for three main metabarcoding markers used in the literature: teleo and MiFish-U/E. We also estimated the number of primer mismatches per species amplified by in silico polymerase chain reaction which influence the probability of species detection. Actinopterygians show a stronger decrease in species richness with depth than Chondrichthyans. These richness gradients are accompanied by a continuous species turnover between depths. Fish species coverage with the MiFish-U/E markers is higher than with teleo while threatened species are more sequenced than the others. "Deep-endemic" species, those not ascending to the shallow depth layer, are less sequenced than not threatened species. The number of primer mismatches is not higher for deep-sea species than for shallower ones. eDNA metabarcoding is promising for species detection in the deep-sea to better account for the 3-dimensional structure of the ocean in marine biodiversity monitoring and conservation. However, we argue that sequencing efforts on "deep-endemic" species are needed.

Benthic invertebrates in Svalbard fjords-when metabarcoding does not outperform traditional biodiversity assessment.

To protect and restore ecosystems and biodiversity is one of the 10 challenges identified by the United Nations's Decade of the Ocean Science. In this study we used eDNA from sediments collected in two fjords of the Svalbard archipelago and compared the taxonomic composition with traditional methods through metabarcoding, targeting mitochondrial CO1, to survey benthos. Clustering of 21.6 mill sequence reads with a d value of 13 in swarm, returned about 25 K OTU reads. An identification search with the BOLD database returned 12,000 taxonomy annotated sequences spanning a similarity range of 50% to 100%. Using an acceptance filter of minimum 90% similarity to the CO1 reference sequence, we found that 74% of the ca 100 taxon identified sequence reads were Polychaeta and 22% Nematoda. Relatively few other benthic invertebrate species were detected. Many of the identified sequence reads were extra-organismal DNA from terrestrial, planktonic, and photic zone sources. For the species rich Polychaeta, we found that, on average, only 20.6% of the species identified from morphology were also detected with DNA. This discrepancy was not due to missing reference sequences in the search database, because 90-100% (mean 96.7%) of the visually identified species at each station were represented with barcodes in Boldsystems. The volume of DNA samples is small compared with the volume searched in visual sorting, and the replicate DNA-samples in sum covered only about 2% of the surface area of a grab. This may considerably reduce the detection rate of species that are not uniformly distributed in the sediments. Along with PCR amplification bias and primer mismatch, this may be an important reason for the limited congruence of species identified with the two approaches. However, metabarcoding also identified 69 additional species that are usually overlooked in visual sample sorting, demonstrating how metabarcoding can complement traditional methodology by detecting additional, less conspicuous groups of organisms.

Genomes of Potato Mop-Top Virus (Virgaviridae: Pomovirus) Isolates from New Zealand and Their Impact on Diagnostic Methods.

Following the detection of potato mop-top virus (PMTV) in New Zealand in 2018, three near-complete PMTV genomes (AS22, AS99, AS144) were assembled from soil samples taken from potato fields in Canterbury. Phylogenetic analysis revealed that these genomes form a distinct lineage, with limited genetic diversity, within the PMTV species. This analysis supports the hypothesis that these genomes share a common origin, possibly resulting from a single (or limited) incursion of PMTV into New Zealand. A single nucleotide polymorphism was identified in the region where a key diagnostic primer binds. The mismatch of the diagnostic primer has implications for the effectiveness of the Mumford diagnostic protocol currently recommended for use in New Zealand; we recommend that the alternative Pandey assay, for which no primer mismatch was detected, be validated and optimized for use on the viral genomes present in New Zealand.

Abstract 3992: Modular ddPCR assay design for highly sensitive single nucleotide variant detection

Abstract Introduction: Single-nucleotide variants (SNVs) are key drivers of both oncogenesis and emergent resistance to targeted therapies. Although high-throughput Identification of SNVs can be achieved with Next Generation Sequencing (NGS) technologies, the cost is significant, and proportional to the desired sensitivity of variant detection. Conversely, while only small numbers of SNVs can be queried in an individual quantitative PCR (qPCR) or droplet digital PCR (ddPCR) reaction, a fully optimized qPCR or ddPCR assay can detect SNVs at very low frequencies for a fraction of the cost of sequencing. Herein, we present a generalizable approach to PCR assay design, using a combination of mutation-sensitive induced mismatch primers, wild-type suppressors, and TaqMan probes, that permits detection of SNVs below 0.1%. Method: Known oncogenic hotspot regions were selected for development of mutation-detecting PCR assays. For each hotspot, a mutation-specific-mismatch primer, suppressor, and probe (MSMPSP) cassette was designed consisting of 1) a mutation-bearing forward primer with engineered mismatches, 2) a wild-type, extension-locked blocker oligo, 3) a fluorescent TaqMan probe, and 4) a downstream reverse primer. Designed mutation assays were evaluated using plasmid control sequences, remnant clinical specimens, and healthy donor peripheral blood samples spiked with known quantities of mutation-bearing plasmid or cell lines and processed on the BioRad AutoDG/QX200 droplet digital PCR system. Results: Variant detection below 0.1% was achieved for all tested mutations, including those residing in homopolymeric and GC-rich regions, from as low as 100 ng DNA input, in a single day. At higher variant frequencies accessible to multiple platforms, results with MSMPSP-based ddPCR assays exhibited excellent concordance with NGS and qPCR results and yielded linear signal decline down to 0.01% variant allele frequency. Overall assay sensitivity was proportional to DNA input, and improved detection limits were achieved by increased DNA input and combined well analysis. Conclusion: Utilizing the MSMPSP approach, coupled with ddPCR, variant detection for targeted mutations below 0.1% is achievable in diverse genomic contexts. Furthermore, simple design principles and flexible probe-binding sites permit rapid development of new assays, allowing critical mutations to be tracked as they are discovered. While multiplexing capabilities are reduced compared with NGS, short time-to-results, coupled with low cost-per-sample for MSMPSP ddPCR, permits faster treatment decisions at reduced cost to the healthcare system. Citation Format: Sarah Heine, Ashley Beams, Nathan Riccitelli, Shabnam Tangri. Modular ddPCR assay design for highly sensitive single nucleotide variant detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3992.

Successes and limitations of quantitative diet metabarcoding in a small, herbivorous mammal.

DNA metabarcoding is widely used to determine wild animal diets, but whether this technique provides accurate, quantitative measurements is still under debate. To test our ability to accurately estimate the abundance of dietary items using metabarcoding, we fed wild-caught desert woodrats (Neotoma lepida) diets consisting of constant amounts of juniper (Juniperus osteosperma, 15%) and varying amounts of creosote (Larrea tridentata, 1%-60%), cactus (Opuntia sp., 0%-100%) and commercial chow (0%-85%). Using metabarcoding, we compared the representation of items in the original diet samples to that in the faecal samples to test the sensitivity and accuracy of diet metabarcoding, the performance of different bioinformatic pipelines and our ability to correct sequence counts. Metabarcoding, using standard trnL primers, detected creosote, juniper and chow. Different pipelines for assigning taxonomy performed similarly. While creosote was detectable at dietary proportions as low as 1%, we failed to detect cactus in most samples, probably due to a primer mismatch. Creosote read counts increased as its proportion in the diet increased, and we could differentiate when creosote was a minor and major component of the diet. However, we found that estimates of juniper and creosote varied. Using previously suggested methods to correct these errors did not improve accuracy estimates of creosote, but did reduce error for juniper and chow. Our results indicate that metabarcoding can provide quantitative information on dietary composition, but may be limited. We suggest that researchers use caution when quantitatively interpreting diet metabarcoding results unless they first experimentally determine the extent of possible biases.

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

MET Exon 14 Skipping Mutations: Essential Considerations for Current Management of Non–Small-Cell Lung Cancer

Disentangling bias for non-destructive insect metabarcoding.

A fast and reliable method for obtaining a species-level identification is a fundamental requirement for a wide range of activities, from plant protection and invasive species management to biodiversity assessments and ecological studies. For insects, novel molecular techniques such as DNA metabarcoding have emerged as a rapid alternative to traditional morphological identification, reducing the dependence on limited taxonomic experts. Until recently, molecular techniques have required a destructive DNA extraction, precluding the possibility of preserving voucher specimens for future studies, or species descriptions. Here we paired insect metabarcoding with two recent non-destructive DNA extraction protocols, to obtain a rapid and high-throughput taxonomic identification of diverse insect taxa while retaining a physical voucher specimen. The aim of this work was to explore how non-destructive extraction protocols impact the semi-quantitative nature of metabarcoding, which alongside species presence/absence also provides a quantitative, but biased, representation of their relative abundances. By using a series of mock communities representing each stage of a typical metabarcoding workflow we were able to determine how different morphological (i.e., insect biomass and exoskeleton hardness) and molecular traits (i.e., primer mismatch and amplicon GC%), interact with different protocol steps to introduce quantitative bias into non-destructive metabarcoding results. We discuss the relevance of taxonomic bias to metabarcoding identification of insects and potential approaches to account for it.

Application of Allele Specific PCR in Identifying Offspring Genotypes of Bi-Allelic SbeIIb Mutant Lines in Rice.

Bi-allelic mutant lines induced by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems are important genetic materials. It is very important to establish a rapid and cheap method in identifying homozygous mutant plants from offspring segregation populations of bi-allelic mutant lines. In this study, the offspring genotypes of rice bi-allelic starch branching enzyme IIb mutant lines were identified using the allele specific PCR (AS-PCR) method. The target sequences of two alleles were aligned from their 5′ to 3′ ends, and the first different bases were used as the 3′ ends of mismatch primers. Another mismatched base was introduced at the third nucleotide from the 3′ end of mismatch primer. The PCR reaction mixture and amplification program were optimized according to the differences of mutation target sequence and mismatch primers. The offspring plant genotypes of bi-allelic mutant lines could be accurately identified using the amplified DNA fragments by agarose gel electrophoresis. This study could provide a method reference for the rapid screening of homozygous mutant plants from offspring segregation population of heterozygous and bi-allelic mutant lines.