Genomic Variations in SARS-CoV-2 Strains at the Target Sequences of Nucleic Acid Amplification Tests

IntroductionNucleic acid amplification is the main method used to detect infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the false-negative rate of nucleic acid tests cannot be ignored.Material and methodsHerein, we demonstrated genomic variations at the target sequences for the tests and the geographical distribution of the variations across countries by analyzing the whole-genome sequencing data of SARS-CoV-2 strains from the 2019 Novel Coronavirus Resource (2019nCoVR) database.ResultsAmong the 21 pairs of primer sequences in regions ORF1ab, S, E, and N, the total length of primer and probe target sequences was 938bp, with 131(13.97%) variant loci in 2415 (38.96%) isolates. Primer targets in the N region contained the most variations that were distributed among the most isolates, and the E region contained the least. Single nucleotide polymorphisms were the most frequent variation, with C to T transitions being detected in the most variant loci. G to A transitions and G to C transversions were the most common and had the highest isolate density. Genomic variations at the three mutation sites N: 28881, N: 28882, and N: 28883 were the most commonly detected, including in 608 SARS-CoV-2 strains from 33 countries, especially in the United Kingdom, Portugal, and Belgium.ConclusionsOur work comprehensively analyzed genomic variations on the target sequences of the nucleic acid amplification tests, offering evidence to optimize primer and probe target sequence selection, thereby improving the performance of the SARS-CoV-2 diagnostic test.

Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching

Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming previously reported problematic aspects of protein interference with graphene oxide. We used a “turn off” assay for specific quenching-based detection of oligo DNA-microRNA hybridization in solution. A fluorescein conjugated 30-mer single-stranded DNA (ssDNA) probe was combined with a complementary synthetic microRNA (18 nucleotides) target. The probe-target hybridization was detected by specific quenching due to photoinduced electron transfer (PET). On the next step, GO captures and quenches the unhybridized probe by fluorescence resonance energy transfer (FRET) in the presence of cell culture medium supplemented with platelet lysate, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100 and 50% formamide. This resulted in sensitive measurement of the specific probe-target complexes remaining in solution. The detection is linear in the range of 1 nM and 8 nM in a single 100 μL total volume assay sample containing 25% cell culture medium supplemented with platelet lysate. We highlight a general approach that may be adopted for microRNA target detection within complex physiological media.

Genomic Variations in SARS-CoV-2 Strains at the Target Sequences of Nucleic Acid Amplification Tests

Background Nucleic acid amplification is the main method used to detect infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the false-negative rate of nucleic acid tests cannot be ignored. Methods Herein, we demonstrated genomic variations at the target sequences for the tests and the geographical distribution of the variations across countries by analyzing the whole-genome sequencing data of SARS-CoV-2 strains from the 2019 Novel Coronavirus Resource (2019nCoVR) database. Results Among the 21 pairs of primer sequences in regions ORF1ab, S, E, and N, the total length of primer and probe target sequences was 938 bp, with 131(13.97%) variant loci in 2415 (38.96%) isolates. Primer targets in the N region contained the most variations that were distributed among the most isolates, and the E region contained the least. Single nucleotide polymorphisms were the most frequent variation, with C to T transitions being detected in the most variant loci. G to A transitions and G to C transversions were the most common and had the highest isolate density. Genomic variations at the three mutation sites N: 28881, N: 28882, and N: 28883 were the most commonly detected, including in 608 SARS-CoV-2 strains from 33 countries, especially in the United Kingdom, Portugal, and Belgium. Conclusions Our work comprehensively analyzed genomic variations on the target sequences of the nucleic acid amplification tests, offering evidence to optimize primer and probe target sequence selection, thereby improving the performance of the SARS-CoV-2 diagnostic test.

Genomic Variations in SARS-CoV-2 Strains at the Target Sequences of Nucleic Acid Amplification Tests

Background: Nucleic acid amplification is the main method used to detect infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the false-negative rate of nucleic acid tests cannot be ignored. Methods: Herein, we demonstrated genomic variations at the target sequences for the tests and the geographical distribution of the variations across countries by analyzing the whole-genome sequencing data of SARS-CoV-2 strains from the 2019 Novel Coronavirus Resource (2019nCoVR) database. Results: Among the 21 pairs of primer sequences in regions ORF1ab, S, E, and N, the total length of primer and probe target sequences was 938bp, with 131(13.97%) variant loci in 2415 (38.96%) isolates. Primer targets in the N region contained the most variations that were distributed among the most isolates, and the E region contained the least. Single nucleotide polymorphisms were the most frequent variation, with C to T transitions being detected in the most variant loci. G to A transitions and G to C transversions were the most common and had the highest isolate density. Genomic variations at the three mutation sites N: 28881, N: 28882, and N: 28883 were the most commonly detected, including in 608 SARS-CoV-2 strains from 33 countries, especially in the United Kingdom, Portugal, and Belgium. Conclusions: Our work comprehensively analyzed genomic variations on the target sequences of the nucleic acid amplification tests, offering evidence to optimize primer and probe target sequence selection, thereby improving the performance of the SARS-CoV-2 diagnostic test.

Identity-Based Crossmodal Negative Priming: Aftereffects of Ignoring in One Sensory Modality on Responding to Another Sensory Modality

Negative Priming (NP) refers to the phenomenon that responses towards previously ignored stimuli, as compared to new stimuli, are impaired. That is, NP is reflected in the performance on the probe display of a prime–probe sequence. NP is established in vision, audition and touch. In the current study, we presented participants with auditory, visual, and tactile manifestations of the same temporal patterns in order to measure NP across the senses. On each trial, the sensory modality shifted from the prime to the probe. Each prime and probe display consisted of a target and a distractor stimulus, presented to the same sensory modality. On some trials, the prime distractor repeated as probe target (ignored-repetition trials), on other trials the probe stimuli had not been involved in the prime display (control trials). We observed NP between audition and touch (Experiment 1) and between vision and audition (Experiment 2). These findings indicate that the processes underpinning NP can operate at an amodal, postperceptual level.

Carbazole modified oligonucleotides: synthesis, hybridization studies and fluorescence properties

Investigation of a modified carbazole as a nucleoside mimetic and its binding affinity towards complementary DNA/RNA models shows improved thermal stability and fluorescence emission of carbazole probe–target hybrid duplexes.