Devices and methods for extraction-free pathogen testing

1 . A method for extraction-free analysis of nucleic acid, the method comprising the steps of:

mixing a combined saliva sample and respiratory mucosa sample in a buffer composition comprising nuclease-free water, an antifungal, an antibiotic, a reducing agent, and a ribonuclease inhibitor;

directly amplifying nucleic acid from the sample in said buffer with primers specific to a target nucleic acid without prior extraction of said nucleic acid; and

analyzing amplicons produced in said amplifying step to detect presence of a pathogen.

2 . The method of claim 1 , wherein said nucleic acid is a pathogen.

3 . The method of claim 2 , wherein said pathogen is a virus or a bacterium.

4 . The method of claim 3 , wherein the virus comprises a coronavirus or an influenza virus.

5 . The method of claim 4 , wherein the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-COV-2).

6 . The method of claim 5 , wherein the method further comprises analyzing the amplicons to detect the presence of one or more target SARS-COV-2 variants.

7 . The method of claim 6 , wherein the method does not include amplification of a wildtype SARS-COV-2 nucleic acid.

8 . The method of claim 4 , wherein the nucleic acid specific primers target one or more of the virus's N, ORF lab, and E genes.

9 . The method of claim 1 , wherein prior to mixing the method comprises obtaining the saliva sample in a vessel and placing a nasal swab comprising the respiratory mucosa sample into the saliva sample in the vessel.

10 . The method of claim 9 , further comprising squeezing and/or agitating the nasal swab in the sample vessel to mix the saliva sample and respiratory mucosa sample.

11 . The method of claim 9 , wherein the nasal swab comprises a respiratory mucosa sample from one or more locations in a nasal cavity.

12 . The method of claim 11 , wherein the one or more locations comprise an anterior nares, a mid-turbinate, and/or a nasopharynx.

13 . The method of claim 12 , wherein the respiratory mucosa sample is obtained from the anterior nares.

14 . The method of claim 1 , wherein said nucleic acid is RNA or DNA.

15 . The method of claim 1 , further comprising the step of comparing nucleic acid quantities in a plurality of combined saliva sample and respiratory mucosa samples obtained from a patient at successive time points and determining disease progression based on increases or decreases in the nucleic acid quantities over time.

16 . The method of claim 15 , further comprising the step of predicting disease outcomes based on the nucleic acid quantity.

17 . A method for extraction-free analysis of nucleic acid, the method comprising the steps of:

providing a vial;

obtaining a saliva sample from a subject in the vial;

obtaining a respiratory mucosa swab sample from the subject;

mixing the saliva sample and respiratory mucosa swab sample in the vial with a buffer composition comprising nuclease-free water, an antifungal, an antibiotic, a reducing agent, and a ribonuclease inhibitor;

directly amplifying nucleic acid from the sample in said buffer with primers specific to a target nucleic acid without prior extraction of said nucleic acid; and

analyzing amplicons produced in said amplifying step to detect presence of a pathogen.

18 . A kit for performing the method of claim 17 , the kit comprising:

the vial;

a saliva collection aid or a funnel;

the buffer composition;

the primers specific for the target nucleic acid; and

instructions for extraction-free analysis of nucleic acid.

19 . A method for stabilizing a viral sample, the method comprising the steps of

obtaining a body fluid sample suspected of containing a virus;

adding to the body fluid sample a buffer comprising nuclease-free water, an antifungal, an antibiotic, a reducing agent, and a ribonuclease inhibitor thereby to stabilize the virus; and

transporting the sample to a testing site.