Granted Patent B2

US 12,264,365 · App. 16/912,462 · Granted Apr 1, 2025

Inventors: Nadya Andini (Livermore, CA); Kathy Chiu (Irvine, CA); Xuewen Jiang (Menlo Park, CA); Hédia Maamar (San Jose, CA)

Assignee: Talis Biomedical Corporation

C12Q1/6876C12Q1/6813C12Q1/6844C12Q1/70C12Q2521/101C12Q2521/107C12Q2531/119C12Q2600/112

Abstract

Claims (28)

1. A composition comprising a set of polynucleotides comprising:

a primer set comprising a forward outer primer comprising SEQ ID NO: 63, a backward outer primer comprising SEQ ID NO: 86, a forward inner primer comprising SEQ ID NO: 65, a backward inner primer comprising SEQ ID NO: 87, a forward loop primer comprising SEQ ID NO: 67, and a backward loop primer comprising SEQ ID NO: 88.

2. The composition of claim 1 , further comprising a probe.

3. The composition of claim 2 , wherein the probe comprises a label.

4. The composition of claim 3 , wherein the probe is a labeled polynucleotide.

5. The composition of claim 4 , wherein the labeled polynucleotide comprises a sequence selected from the group consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79.

6. The composition of claim 5 , wherein the labeled polynucleotide comprises SEQ ID NO.: 77 or SEQ ID NO.: 79.

7. The composition of claim 5 , wherein the sequence of the labeled polynucleotides is SEQ ID NO.: 77.

8. The composition of claim 2 , wherein the probe is a molecular beacon comprising a fluorophore, a quencher, and a polynucleotide.

9. The composition of claim 8 , wherein the molecular beacon comprises a sequence selected from the group consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79.

10. The composition of claim 9 , wherein the molecular beacon comprises a sequence selected from the group consisting of SEQ ID NO.: 77 and SEQ ID NO.: 79.

11. A kit comprising a composition according to claim 1 .

12. The kit of claim 11 , further comprising a strand displacement polymerase and reverse transcriptase.

13. The kit of claim 12 , further comprising a molecular beacon comprising a fluorophore, a quencher, and a polynucleotide, wherein the polynucleotide comprises a sequence selected from the group consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79.

14. The kit of claim 13 , wherein the polynucleotide of the molecular beacon consists of a sequence selected from the group consisting of SEQ ID NO.: 77 and SEQ ID NO.: 79.

15. A method of detecting SARS-CoV-2 in a test sample, the method comprising:

(a) extracting nucleic acid from the test sample;

(b) amplifying a target sequence by reacting the nucleic acid extracted in step (a) with a reaction mixture comprising a strand displacement DNA polymerase and reverse transcriptase and a sequence-specific primer set, wherein said sequence-specific primer set is Set-13, wherein Set-13 comprises a forward outer primer comprising SEQ ID NO: 63, a backward outer primer comprising SEQ ID NO: 86, a forward inner primer comprising SEQ ID NO: 65, a backward inner primer comprising SEQ ID NO: 87, a forward loop primer comprising SEQ ID NO: 67, and a backward loop primer comprising SEQ ID NO: 88; and

(c) detecting the presence or absence of an amplified product of step (b); wherein the presence of said amplification product is indicative of the presence of SARS-CoV-2 in the test sample.

16. The method of claim 15 , wherein the amplification in step (b) of the target sequence is performed between about 60° C. and about 67° C. for less than 30 minutes.

17. The method of claim 16 , wherein the amplification step is performed for less than fifteen minutes.

18. The method of claim 17 , wherein the amplification step is performed for less than twelve minutes.

19. The method of claim 18 , wherein the amplification step is performed for less than nine minutes.

20. The method of claim 15 , wherein detecting the presence or absence of the amplification product comprises hybridizing the amplified product with a probe comprising a polynucleotide attached to a label.

21. The method of claim 20 , wherein the label is a fluorophore.

22. The method of claim 21 , wherein the fluorophore is covalently attached to a terminus of the polynucleotide.

23. The method of claim 20 , wherein the labeled polynucleotide comprises a sequence selected from the group consisting of nucleotides 5-29 of SEQ ID NO.: 77 and nucleotides 5-29 of SEQ ID NO.: 79.

24. The method of claim 23 , wherein the sequence of the labeled polynucleotide comprises SEQ ID NO.: 77 or SEQ ID NO.: 79.

Assignments (5)

Continuity (3)

References Cited (187)