Massively scalable viral testing and asymptomatic surveillance

A method of rapid identification of SARS-CoV-2 positive patient(s) within a group comprising a plurality of patients in need of evaluation for SARS-CoV-2 infection comprising:

• (a) separately incubating RNA-containing samples obtained from each of the plurality of patients with an oligonucleotide comprising a patient-specific barcode, wherein the oligonucleotide comprises a sequence complementary to a SARS-CoV-2 target sequence, under conditions in which, if an RNA-containing sample comprise SARS-CoV-2 genomic RNA, an oligonucleotide-SARS-CoV-2 RNA complex is produced comprising the oligonucleotide hybridized to the SARS-CoV-2 RNA,
• thereby producing a plurality of incubated patient samples each of which comprises an oligonucleotide with a different patient-specific identifying barcode sequence
• (b) pooling the plurality of incubated patient samples to produce a pooled sample;
• (c) purifying oligonucleotide-SARS-CoV-2 RNA complexes, when present, from the pooled sample;
• (d) performing a reverse transcriptase reaction to extend the oligonucleotide hybridized to the SAR-Co-V-2 RNA in the oligonucleotide-SARS-CoV-2 RNA complexes;
• and
• (e) amplifying the ligation products, if present, to produce amplicons;
• and
• (f) detecting the amplicons,
• wherein detection of amplicons in a pooled sample indicates that one or more of the patient samples comprises SARS-CoV-2 RNA and one or more of the patients is positive for the presence of SARS-CoV-2.

A method of rapid identification of single-stranded RNA (ssRNA) virus-positive patient(s) within a group comprising a plurality of patients in need of evaluation for infection with a ssRNA virus, comprising:

• (a) separately incubating RNA-containing samples obtained from each of the plurality of patient with three or more collinear oligonucleotides,
• wherein at least one of the three or more collinear oligonucleotides comprises a patient-specific identifying barcode sequence,
• wherein the three or more collinear oligonucleotides have sequences complementary to a ssRNA virus target sequence, under conditions in which, if an RNA-containing sample comprises ssRNA virus genomic RNA, an oligonucleotide-viral RNA complex is produced comprising the three or more collinear oligonucleotides hybridized at adjacent positions in the ssRNA virus RNA target sequence,
• thereby producing a plurality of incubated patient samples each of which comprises an oligonucleotide with a different patient-specific identifying barcode sequence, and
• (b) pooling the plurality of incubated patient samples to produce a pooled sample;
• (c) purifying oligonucleotide-viral RNA complexes, when present, from the pooled sample;
• (d) ligating the three or more collinear oligonucleotides hybridized to viral RNA in the oligonucleotide-viral RNA complexes, if present, to produce ligation products comprising patient-specific identifying barcode sequences, and
• (e) amplifying the ligation products, if present, to produce amplicons,
• (f) detecting the amplicons,
• wherein detection of amplicons in a pooled sample indicates that one or more of the patient samples comprises ssRNA virus RNA and one or more of the patients is positive for infection with the ssRNA virus.

A method of rapid identification of ssRNA virus-positive patient(s) within a group comprising a plurality of patients in need of evaluation for infection with a ssRNA virus, comprising:

• (a) separately incubating RNA-containing samples obtained from each of the plurality of patients with an oligonucleotide comprising a patient-specific barcode, wherein the oligonucleotide comprises a sequence complementary to a ssRNA virus target sequence, under conditions in which, if an RNA-containing sample comprises ssRNA virus genomic RNA, an oligonucleotide-viral RNA complex is produced comprising the oligonucleotide hybridized to the viral RNA,
• thereby producing a plurality of incubated patient samples each of which comprises an oligonucleotide with a different patient-specific identifying barcode sequence;
• (b) pooling the plurality of incubated patient samples to produce a pooled sample;
• (c) purifying oligonucleotide-viral RNA complexes, when present, from the pooled sample;
• (d) performing a reverse transcriptase reaction to extend the oligonucleotide hybridized to the viral RNA in the oligonucleotide-viral RNA complexes;
• and
• (e) amplifying the ligation products, if present, to produce amplicons;
• and
• (f) detecting the amplicons,
• wherein detection of amplicons in a pooled sample indicates that one or more of the patient samples comprises ssRNA virus RNA and one or more of the patients is positive for the presence of ssRNA virus.