PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs

Monitoring diseases caused by pathogens or by mutations in DNA sequences requires accurate, rapid, and sensitive tools to detect specific nucleic acid sequences. Here, we describe a new peptide nucleic acid (PNA)-based nucleic acid detection toolkit, termed PNA-powered diagnostics (PNA-Pdx). PNA-Pdx employs PNA probes that bind specifically to a target and are then detected in lateral flow assays. This can precisely detect a specific pathogen or genotype genomic sequence. PNA probes can also be designed to invade double-stranded DNAs (dsDNAs) to produce single-stranded DNAs for precise CRISPR-Cas12b-based detection of genomic SNPs without requiring the protospacer-adjacent motif (PAM), as Cas12b requires PAM sequences only for dsDNA targets. PNA-Pdx identified target nucleic acid sequences at concentrations as low as 2 copies/μL and precisely detected the SARS-CoV-2 genome in clinical samples in 40 min. Furthermore, the specific dsDNA invasion by the PNA coupled with CRISPR-Cas12b precisely detected genomic SNPs without PAM restriction. Overall, PNA-Pdx provides a novel toolkit for nucleic acid and SNP detection as well as highlights the benefits of engineering PNA probes for detecting nucleic acids

Additional file 1: Figure S1. of RNA virus interference via CRISPR/Cas13a system in plants

Confirmation of pCas13a expression in planta. Figure S2. pCas13a-mediated interference with TuMV-GFP in planta. Figure S3. GFP quantification of TuMV-GFP interference in transgenic pCas13a-OE plants. (PDF 1393 kb

Additional file 2: Sequence S1: of RNA virus interference via CRISPR/Cas13a system in plants

pCas13a amino acid sequence (3xHA-pCas13a-nls). Sequence S2: pCas13a full-length plant codon optimized DNA sequence (3x-HA-pCas13a-nls) Map S1: pCas13a sequence in pK2GW7-pCas13a (pK2GW7-3xHA-pCas13a-nls). Sequence S3: TuMV-GFP full-length sequence with target sequences in different colors. Map S2: Map of TuMV-GFP. Sequence S4: Cas13a-repeat-cRNA-TuMV-GFP-GFP-target 1 sequence (A), map (B) and its complex with targeting region in TuMV GFP genomic region (C). Sequence S5: Cas13a-repeat-cRNA-TuMV-GFP-GFP-T2 sequence (A), map (B), and its complex with targeting area in TuMV GFP genomic region (C). Sequence S6: Cas13a-repeat-cRNA-TuMV-GFP-HC-Pro-T1 sequence (A), map (B), and its complex with HC-pro targeting region in TuMV (C). Sequence S7: Cas13a-repeat-cRNA-TuMV-GFP-Cp-Pro-T1 sequence (A), map (B), and its complex with Capsid protein targeting region in TuMV. (DOCX 414 kb

Additional file 3: Table S1: of RNA virus interference via CRISPR/Cas13a system in plants

TuMV genome description. Table S2: Primers used in this study. (DOCX 21 kb

CD4 Counts across various categories of heroin injectors.

<p>CD4 T-cells are compared between treatment arms of injectors (A). ¶ Counts are significantly lower for the D4T- arm than for the AZT arm (p = 0.004), TDF (p = 0.005) or the ART- (p<0.001) arm and lower for the sub-optimal ART than ART- (p = 0.023) arm. All subjects in the ‘ART-’ arm were not infected (NI) by either virus. Mean CD4 counts are compared between infection statuses (B). These are significantly lower for co-infected than HIV-1 mono infected injectors as shown. §Shows significantly lower CD4 counts for HIV mono-infected than NI injectors (p = 0.002). CD4 data is compared between age groups of the different infection statuses (C). No significant (NS) difference was observed. *Co-infected injectors had much lower CD4 levels compared to other infection categories in any age group. Only one injector was HCV mono-infected (horizontal bar in the >30–40 years category). CD4 Counts were not significantly different between genders of injectors (D). ART, antiretroviral treatment; CTX, cotrimoxazole (septrin); ART-, No ART; Uk, unknown ART status; Sub, sub optimal ART.</p

CD4 T-cell counts compared by various categories of heroin injectors who were screened for both HIV and HCV.

<p>Legend of table:</p><p>^† P-value is significant at level shown comparing mean CD4 between infection statuses or between treatment arms.</p><p>^a No qualifying subjects.</p><p>^b Confidence Interval (CI) is not applicable. ART, antiretroviral treatment. Only one subject (aged 31-40yrs, CD4 T-cells of 287 counts/mm³) was HCV mono-infected, and is excluded from this table. Sub-optimal ART cases are IHUs with unexplained use of single-drug ART regimen.</p><p>CD4 T-cell counts compared by various categories of heroin injectors who were screened for both HIV and HCV.</p