Methods and reagents for rapid detection of pathogens in biological samples
Methods and compositions for rapid detection of pathogens in biological samples, such as saliva or a nasal swab, are disclosed. Nucleic acid molecules from pathogens can be stabilized at room temperature for several days in a transport solution, followed by their amplification without prior extraction. An optimized isothermal amplification method and the amplification solution are also disclosed, allowing for reducing a threshold time for a positive sample. The methods and compositions are suitable for detection of a variety of pathogens, including enveloped viruses, such as Cytomegalovirus or SARS-CoV-2 virus, and pathogenic microorganisms.
1 . A method for amplifying a nucleic acid molecule of a pathogen contained in a biological sample in less than 30 min, the method comprising:
(a) obtaining a stabilized sample, the stabilized sample comprising the biological sample and a transport solution, wherein the transport solution comprises a non-ionic or zwitterionic detergent at a concentration of 2 to 32 percent (vol./vol.), and wherein the transport solution stabilizes the nucleic acid molecule in the stabilized sample at room temperature;
(b) mixing the stabilized sample with an amplification solution without prior extraction of the nucleic acid molecule present in the stabilized sample to form an amplification reaction mixture, wherein the amplification reaction mixture comprises:
a buffering agent that has a pH between about 5 and about 11;
serum albumin at a concentration of 100 μM to 500 μM;
the non-ionic or zwitterionic detergent at a concentration of 1 to 16 percent (vol./vol.);
M 2 SO 4 at a concentration of 0.5 mM to 3 mM:
dNTPs, a DNA polymerase enzyme having a strand displacement activity selected from the group consisting of Bst polymerase and Bsm polymerase, and at least one pair of primers that target the nucleic acid molecule; and
(c) subjecting the amplification reaction mixture to an isothermal amplification, wherein the isothermal amplification amplifies the nucleic acid molecule.
2 . The method of claim 1 , wherein the transport solution further comprises a buffering agent that has a pH between about 5 and about 11, and a chelating agent.
3 . The method of claim 1 , wherein the transport solution stabilizes the nucleic acid in the stabilized sample at room temperature for at least four weeks.
4 . The method of claim 1 , wherein the pathogen is selected from the group consisting of: an enveloped virus, pathogenic yeast and bacteria.
5 . The method of claim 1 , wherein the amplification reaction mixture comprises MgSO4 at a concentration of 2 mM.
6 . The method of claim 1 , wherein the amplification reaction mixture comprises at least two pairs of primers that target the nucleic acid molecule, and the isothermal amplification is a strand displacement amplification, a multiple displacement amplification, a recombinase polymerase amplification, a helicase dependent amplification, a rolling circle amplification, or a loop-mediated isothermal amplification.
7 . The method of claim 1 , wherein the nucleic acid molecule is a ribonucleic acid (RNA) and a corresponding DNA molecule is produced from the RNA by a reverse transcriptase enzyme prior to or simultaneously with the isothermal amplification.
8 . The method of claim 1 , wherein the biological sample is saliva, a nasal swab containing the pathogen, urine, lesion exudate, dried blood spot, blood, plasma/serum, mucus, vaginal fluid or another type of bodily fluid.
9 . The method of claim 1 , wherein the transport solution consists essentially of the non-ionic or zwitterionic detergent at a concentration of 2 to 32 percent (vol./vol.).
10 . The method of claim 1 , wherein the non-ionic or zwitterionic detergent is selected from the group consisting of: octylphenol ethoxylate, nonylphenol ethoxylate, octylphenol ethoxylate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, and a mixture thereof.
11 . The method of claim 1 , wherein the non-ionic or zwitterionic detergent is present in the amplification reaction mixture at a concentration of 2 to 5 percent (vol./vol.).
12 . A method for detecting a presence of a pathogen in a biological sample in less than 30 min, comprising:
(a) obtaining a stabilized sample, the stabilized sample comprising the biological sample and a transport solution, wherein the transport solution comprises a non-ionic or zwitterionic detergent at a concentration of 2 to 32 percent (vol./vol.), and wherein the transport solution stabilizes the nucleic acid molecule in the stabilized sample at room temperature;
(b) mixing the stabilized sample with an amplification solution without prior extraction of the nucleic acid molecule present in the stabilized sample to form an amplification reaction mixture, wherein the amplification reaction mixture comprises:
a buffering agent that has a pH between about 5 and about 11;
serum albumin at a concentration of 100 μM to 500 μM;
the non-ionic or zwitterionic detergent at a concentration of 1 to 16 percent (vol./vol.);
MgSO 4 at a concentration of 0.5 mM to 3 mM;
dNTPs, a DNA polymerase enzyme having a strand displacement activity selected from the group consisting of Bst polymerase and Bsm polymerase, and at least one pair of primers that target the nucleic acid molecule; and
(c) subjecting the amplification reaction mixture to an isothermal amplification, wherein the isothermal amplification amplifies the nucleic acid molecule of the pathogen; and
(d) detecting a presence of the amplified nucleic acid molecule by at least one of the following: colorimetric readout, fluorescent readout, lateral flow assay, a biosensor, wherein the presence of the amplified nucleic acid molecule indicates the presence of the pathogen in the biological sample.
13 . A method for amplifying a nucleic acid molecule of a pathogen contained in a biological sample in less than 30 min, the method comprising:
(a) obtaining a stabilized sample, the stabilized sample consisting essentially of the biological sample and a transport solution, wherein the transport solution consists essentially of a non-ionic or zwitterionic detergent at a concentration of 2 to 32 percent (vol./vol.), and wherein the transport solution stabilizes the nucleic acid molecule in the stabilized sample at room temperature;
(b) mixing the stabilized sample with an amplification solution without prior extraction of the nucleic acid molecule present in the stabilized sample to form an amplification reaction mixture comprising the non-ionic or zwitterionic detergent at a concentration of 1 to 16 percent (vol./vol.), MgSO 4 at a concentration of 0.5 mM to 3 mM and Bst polymerase or Bsm polymerase; and
(c) subjecting the amplification reaction mixture to an isothermal amplification, wherein the isothermal amplification amplifies the nucleic acid molecule.
14 . The method of claim 13 , wherein the non-ionic or zwitterionic detergent is selected from the group consisting of: octylphenol ethoxylate, nonylphenol ethoxylate, octylphenol ethoxylate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate f-HAP-S, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, and a mixture thereof.
15 . The method of claim 13 , wherein the transport solution stabilizes the nucleic acid in the stabilized sample at room temperature for at least four weeks.
16 . The method of claim 13 , wherein the nucleic acid molecule is a ribonucleic acid (RNA) and a corresponding DNA molecule is produced from the RNA by adding a reverse transcriptase enzyme prior to or simultaneously with the step (c).
17 . The method of claim 13 , wherein the non-ionic or zwitterionic detergent is present in the amplification reaction mixture at a concentration of 2 to 5 percent (vol./vol.).