Reagents and oligonucleotides used are listed in Tables 1 and and2.2. The molecular beacons were synthesized using a K&A H-8 SE DNA synthesizer and purified by reverse-phase high-performance liquid chromatography (ATDBio). Molecular beacons are reconstituted with annealing buffer [10 mM tris (pH 8) with 10 μM MgCl2] to the final concentration of 10 μM and then annealed by incubation at 85°C for 5 min and then gradual cooling to 4°C by 0.1°C/s before the NASBA reaction.

DTT, dithiothreitol; DMSO, dimethyl sulfoxide; dNTP, deoxynucleotide triphosphate; NTP, nucleotide triphosphate; BSA, bovine serum albumin; HS, high sensitivity; dsDNA, double-stranded DNA; qPCR, quantitative PCR; NEB, New England Biolabs.

Mix crude saliva (commercial pooled human saliva from healthy individuals) at 1:1 ratio with QuickExtract DNA Extraction Solution. Incubate at 95°C for 5 min to ensure complete lysis of virus and inactivation of proteinase K.

Take 1 μl from the product of step 1 (saliva lysate) and add into the NASBA reaction mixture (without the enzyme mix) to make a total volume of 15 μl. Reaction mixture can either be prepared in-house or from the Life Sciences NASBA liquid kit (see Table 3 below) using one of the two temperature settings below.

*See table S1 for detailed composition.

1) Reaction mixture without the enzyme mix is incubated at 65°C for 2 min, followed by a 10-min incubation at 41°C. Following that, 5 μl of enzyme mix is added into the reaction and incubated at 41°C for a further of 90 to 120 min.

2) Alternatively, reaction mixture without the enzyme mix is incubated at 95°C for 5 min, followed by a 10-min incubation at 41°C. Following incubation, 5 μl of enzyme mix is added into the reaction and incubated at 41°C for a further 90 to 120 min.

A fluorescence plate reader (e.g., FLUOstar) can be used to monitor the reaction in real-time or as an end point assay.

For detection with a lateral flow assay, a NASBA-lyophilized kit is used with the constitution of the reaction mixture shown in Table 4. Take 4 μl from the product of step 1 (saliva lysate) and add into the NASBA reaction mixture (without the enzyme mix) to make a total volume of 60μl. Incubate at 95°C for 5 min, followed by a 10-min incubation at 41°C.

Following that, 20 μl of enzyme mix is added into the reaction and incubated at 41°C for a further of 90 to 120 min. Take the reaction product to the sample well of a PCRD test cassette. Results will be shown within 10 min.

To allow for pooled sequencing of NASBA reaction end products, barcode sequences are added upstream of each of the forward and reverse primers (Fig. 4A). In addition, an Illumina sequencing adaptor is added upstream of the forward primer barcode sequence as a universal PCR handle (see Table 2 for the oligonucleotide sequences).

NASBA end products (2 μl) from each sample are first pooled into a single tube. Pooled products are then column purified to remove residual NASBA primers (QIAquick PCR Purification Kit). PCR is performed on the column-purified pooled sample using two NGS indexing primers and the reaction mix and cycling parameters in Table 5. Here, we have designed a customized NGS primer containing the T7 polymerase promoter sequence (see Table 2 for the oligonucleotide sequence) at the P5 end and used a standard TruSeq sequencing primer at the P7 side. A PCR mix is made on the basis of Table 5 below. A standard PCR program is used with longer elongation time and minimal cycle number to reduce barcode hopping.

After the PCR, an AMPure bead–based double size selection is carried out (0.55× and 0.75×) to enrich for products of interest. In this study, NGS was carried out using 150–bp (base pair) paired-end MiSeq sequencing with MiSeq Reagent Kit v2 (300 cycles). Before sequencing, 30% PhiX was added into the library to increase the complexity.

To analyze the INSIGHT NGS data, sequences in FASTQ files are first trimmed to leave the first 80 nucleotides for both read 1 and read 2 using FASTX_trimmer. The trimmed read 1 and paired read 2 are then merged by FLASH. The merged sequence is compared with the 102-nt reference viral genome sequence (NNNNNACACCTGTGCCTGTTAAACCATTGAAGTTGAAATTGACACATTTGTTTTTAACCAAATTAGTAGACTTTTTAGGTCCACAAACAGTTGCTGGNNNNN, where N stands for the barcode position), and only those with a Hamming distance of less than or equal to 2 are extracted. Here, only substitutions were allowed, while insertion- and deletion-containing reads were filtered out. The first 5-nt and the final 5-nt regions of all extracted sequences correspond, respectively, to the right barcode and the reverse complement of the left barcode. Diagnostic results for sequenced NASBA samples are determined according to the read counts of their corresponding sample-specific barcode pairs (only sequences with exact barcode match were counted). More details can be found in Results.

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