DETECTING A TARGET NUCLEIC ACID IN A BIOLOGICAL SAMPLE
Provided herein are methods, compositions, and kits for detecting a target nucleic acid, such as from a virus, in a biological sample. More specifically, the methods, compositions, and kits described herein describe detection of target nucleic acid from a coronavirus, such as SARS-CoV- 2 coronavirus, with non-ionic detergents and isothermal amplification.
1 . A method for detecting a presence of a target nucleic acid in a biological sample, the method comprising:
creating a mixture in a container, the mixture comprising:
the biological sample,
a non-ionic detergent,
one or more primers for specifically binding to the target nucleic acid in the biological sample, or a complement thereof,
one or more probes for the target nucleic acid, and
one or more polymerases,
wherein the mixture is created prior to subjecting the target nucleic acid to a nucleic acid extraction or lysis;
incubating the mixture to react the non-ionic detergent with the biological sample;
amplifying the target nucleic acid, or a complement thereof, by polymerization using the one or more polymerases to generate an amplified target nucleic acid product, or a complement thereof; and
detecting the amplified target nucleic acid product or complement thereof with the one or more probes, thereby detecting the presence of the target nucleic acid in the biological sample.
2 . The method of claim 1 , wherein the non-ionic detergent is selected from a group consisting of: Tween 20, Tween 80, Triton X-100, NP 40, ECOSURF™ SA, Brij-58, and combinations thereof.
3 . The method of claim 2 , wherein the non-ionic detergent is present at a concentration from about 0.01% to about 10.0%.
4 . The method of claim 1 , wherein the biological sample is lysed for a period of time from about 30 seconds to about 20 minutes at a temperature from about 35° C. to about 75° C.
5 . The method of claim 1 , further comprising contacting the mixture with an RNase inhibitor and/or a uracil-DNA glycosylase.
6 . The method of claim 1 , wherein the target nucleic acid is a viral nucleic acid comprising DNA.
7 . The method of claim 1 , wherein the target nucleic acid is a viral nucleic acid comprising RNA.
8 . The method of claim 7 , wherein the viral nucleic acid is reverse transcribed using a reverse transcriptase selected from a group consisting of: MMLV, MMLV (RNase H minus), SuperScript II, SuperScript III, SuperScript IV, RevertAid H Minus, Maxima H, ProtoScript II, EnzScript™, ABscript II, EpiScript™, or RocketScript (Bioneer), and combinations thereof two or more times at a temperature of about 50° C. to about 70° C.
9 . The method of claim 7 , wherein the viral nucleic acid comprises viral nucleic acid from a bacteriophage, wherein the bacteriophage is an MS2 bacteriophage.
10 . The method of claim 9 , further comprising detecting a control nucleic acid, wherein the control nucleic acid is a MS2 bacteriophage gene.
11 . The method of claim 7 , wherein the viral nucleic acid comprises viral nucleic acid from a coronavirus and, optionally, wherein the coronavirus comprises a SARS-CoV-2 virus.
12 . The method of claim 11 , further comprising detecting the SARS-CoV-2 virus in the biological sample, wherein detecting the SARS-CoV-2 virus in the biological sample comprises detecting a SARS-CoV2 N gene and/or a SARS-CoV-2 ORF gene.
13 . The method of claim 1 , wherein the biological sample is obtained from a human, and optionally, wherein diagnosing the human with COVID-19 disease comprises detecting the SARS-CoV-2 virus in the human biological sample.
14 . The method of claim 1 , further comprising isothermally amplifying the target nucleic acid, wherein isothermally amplifying comprises one of a helicase-dependent amplification, a loop mediated isothermal amplification, a recombinase polymerase amplification, or a rolling circle amplification.
15 . The method of claim 1 , wherein the one or more polymerases comprises a DNA polymerase.
16 . The method of claim 1 , wherein the amplified target nucleic acid product is detected using a quantitative PCR method with the one or more probes.
17 . The method of claim 16 , wherein the quantitative PCR method comprises a real-time PCR assay and, optionally, wherein the quantitative PCR method comprises a TaqMan™ assay.
18 . The method of claim 16 , wherein the quantitative PCR method employs a non-sequence-specific double-stranded DNA-binding dye to detect the amplified target nucleic acid product and wherein the non-sequence specific double-stranded DNA binding dye is SYBR green.
19 . A method for detecting the presence of a viral nucleic acid in a biological sample, the method comprising:
incubating a non-ionic detergent with the biological sample to react the non-ionic detergent with the biological sample;
prior to subjecting the viral nucleic acid to a nucleic acid extraction or lysis, contacting the biological sample and the non-ionic detergent with a mixture comprising:
one or more primers for specifically binding to the viral nucleic acid in the biological sample, or a complement thereof,
one or more probes for the viral nucleic acid, and
one or more polymerases;
amplifying the viral nucleic acid, or a complement thereof, by polymerization using the one or more polymerases to generate an amplified viral nucleic acid product, or a complement thereof; and
detecting the amplified viral nucleic acid product, or complement thereof, with the one or more probes, thereby detecting the presence of the virus in the biological sample.
20 . A kit comprising:
(i) one or more polymerases;
(ii) one or more primers for a viral nucleic acid;
(iii) a non-ionic detergent;
(iv) one or more probes; and
(v) instructions for creating a mixture comprising a biological sample containing the viral nucleic acid, the one or more polymerases, the one or primers, the non-ionic detergent, and the one or more probes, wherein the mixture is created prior to subjecting the viral nucleic acid to a nucleic acid extraction or a lysis.