Unique Barcoded Primer-Assisted Sample-Specific Pooled Testing (Uni-Pool) for Large-Scale Screening of Viral Pathogens.

Pooled testing has been widely adopted recently to facilitate large-scale community testing during the COVID-19 pandemic. This strategy allows to collect and screen multiple specimen samples in a single test, thus immensely saving the assay time and consumable expenses. Nevertheless, when the outcome of a pooled testing is positive, it necessitates repetitive retesting steps for each sample which can pose a serious challenge during a rising infection wave of increasing prevalence. In this work, we develop a unique barcoded primer-assisted sample-specific pooled testing strategy (Uni-Pool) where the key genetic sequences of the viral pathogen in a crude sample are extracted and amplified with concurrent tagging of sample-specific identifiers. This new process improves the existing pooled testing by eliminating the need for retesting and allowing the test results-positive or negative-for all samples in the pool to be revealed by multiplex melting curve analysis right after real-time polymerase chain reaction. It significantly reduces the total assay time for large-scale screening without compromising the specificity and detection sensitivity caused by the sample dilution of pooling. Our method was able to successfully differentiate five samples, positive and negative, in one pool with negligible cross-reactivity among the positive and negative samples. A pooling of 40 simulated samples containing severe acute respiratory syndrome coronavirus-2 pseudovirus of different loads (min: 10 copies/µL; max: 103 copies/µL) spiked into artificial saliva was demonstrated in eight randomized pools. The outcome of five samples in one pool with a hypothetical infection prevalence of 15% in 40 samples was successfully tested and validated by a typical Dorman-based pooling.

Kinetically-enhanced DNA detection via multiple-pass exonuclease III-aided target recycling.

One of the promising approaches to address the challenge of detecting dilute nucleic acid analytes is exonuclease III-aided target recycling. In this strategy, the target DNA self-assembles with the reactant DNA probes and displays itself as a reactant and product at the same time. This provides an autonomous mechanism to release and reuse the analyte from each round of reactions for repetitive cycles, which amplifies the signal without amplifying the analyte itself. However, for very low amounts of the analyte, it takes a considerably long time before a detectable signal is generated. Thus, in this paper, we report a kinetically-enhanced target recycling strategy by designing two more target recycling sub-reactions that are triggered by the byproducts of the first reaction involving the target analyte. In this manner, concentrations of up to 0.5 pM of target DNA can be detected in 15 minutes.

Prostate cancer detection through unbiased capture of methylated cell-free DNA.

Prostate cancer screening using prostate-specific antigen (PSA) has been shown to reduce mortality but with substantial overdiagnosis, leading to unnecessary biopsies. The identification of a highly specific biomarker using liquid biopsies, represents an unmet need in the diagnostic pathway for prostate cancer. In this study, we employed a method that enriches for methylated cell-free DNA fragments coupled with a machine learning algorithm which enabled the detection of metastatic and localized cancers with AUCs of 0.96 and 0.74, respectively. The model also detected 51.8% (14/27) of localized and 88.7% (79/89) of patients with metastatic cancer in an external dataset. Furthermore, we show that the differentially methylated regions reflect epigenetic and transcriptomic changes at the tissue level. Notably, these regions are significantly enriched for biologically relevant pathways associated with the regulation of cellular proliferation and TGF-beta signaling. This demonstrates the potential of circulating tumor DNA methylation for prostate cancer detection and prognostication.

Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity.

The sensitive and accurate detection of rare mutations has profound clinical implications; however, current methods require expensive instrumentation and are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations. Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection but its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. Here, we report an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans-cleavage activity. We show that our strategy can allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.

Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands.

The arrangement of multiple single nucleotide polymorphisms (SNPs) in a gene, called a haplotype phase, is increasingly recognized as critical for accurate determination of disease risk and severity. However, conventional toehold-mediated strand displacement reactions are only able to interrogate SNPs, but not phase them since it is not known whether two SNPs in the same copy of the gene (cis) or in different copies of the same gene (trans) will give the same readout. While the rational introduction of an enzyme enables haplotype phasing, the complicated and stable secondary structure of long, single-stranded DNA sequences at room temperature limits its use. Complex nucleic acid structures make the hybridization of the probes difficult. Thus, we designed a molecular method to reveal the relative positions of SNPs located 1.4 kb apart in two copies of a gene by employing a competitive toehold probes and sink strategy at an elevated temperature. As such, we have successfully differentiated 20 nM of the 10 possible diplotypes in a long DNA target with two SNP sites located 1.4 kb apart within an hour without any additional amplification step. This offers a promising technology for accurate and fast haplotype phasing of SNPs that are over multiple kilobases away from each other.

Oligonucleotide hybridization with magnetic separation assay for multiple SNP phasing.

Since humans have two copies of each gene, multiple mutations in different loci may or may not be found on the same strand of DNA (i.e., inherited from one parent). When a person is heterozygous at more than one position, the placement of these mutations, also called the haplotype phase, (i.e., cis for the same strand and trans for different strands) can result in the expression of different amount and type of proteins. In this work, we described an enzyme-free method to phase two single nucleotide polymorphisms (SNPs) using two fluorophore/quencher-labelled probes, where one of which was biotinylated. The fluorescence signal was obtained twice: first, after the addition of the labelled probes and second, after the addition of the magnetic beads. The first signal was shown to be proportional to the total number of SNP A and SNP B present in the target analyte, while the second signal showed a marked decrease of the fluorescence signal from the non-biotinylated probe when the SNPs were in trans, showing that the probe immobilized on the magnetic bead selectively captures targets with SNPs in a cis configuration. We then mimic the nature of the human genome which consists of two haplotype copies of each gene, and showed that 250 nM of the 10 possible pairs of haplotypes could be differentiated using a combination of fluorescence microscopy and fluorescence detection.