DETECTION OF NUCLEIC ACIDS IN CRUDE MATRICES
A method includes contacting a crude matrix with components of an isothermal nucleic acid amplification reaction for a target nucleic acid species, thereby providing a mixture; incubating the mixture under conditions sufficient for the isothermal nucleic acid amplification reaction to proceed, thereby providing a product; and determining whether an indicator of the target nucleic acid species is present in the product.
1 - 54 . (canceled)
55 . A method, comprising:
performing an isothermal nucleic acid amplification reaction of a mixture to provide a product, the mixture comprising a crude matrix and components of an isothermal nucleic acid amplification reaction for a target nucleic acid species; and
determining whether an indicator of the target nucleic acid species is present in the product.
56 . The method of claim 55 , wherein the method comprises:
contacting the crude matrix with the components of the isothermal nucleic acid amplification reaction for the target nucleic acid species to form the mixture; and
incubating the mixture under conditions sufficient for the isothermal nucleic acid amplification reaction to proceed.
57 . The method of claim 55 , wherein the method comprises:
contacting the crude matrix with the components of the isothermal nucleic acid amplification reaction for the target nucleic acid species to form the mixture; and
maintaining the mixture at a temperature of less than 80° C. for a time sufficient for the isothermal nucleic acid amplification reaction to proceed.
58 . The method of claim 55 , wherein the method comprises:
contacting the crude matrix with the components of the isothermal nucleic acid amplification reaction for the target nucleic acid species to form the mixture; and
varying a Celsius-scale temperature of the mixture by less than 25% or 15° C. for a time sufficient to allow the isothermal nucleic acid amplification reaction to proceed.
59 . The method of claim 55 , wherein the method comprises incubating the mixture at a temperature of at most 80° C. to provide a product.
60 . The method of claim 55 , wherein the method comprises incubating the mixture while varying a Celsius-scale temperature of the mixture by at most 25% or 15° C. to provide a product.
61 . The method of claim 55 , wherein the crude matrix is a biological sample.
62 . The method of claim 61 , wherein the biological sample comprises at least one component selected from the group consisting of blood, urine, saliva, sputum, lymph, plasma, ejaculate, lung aspirate, and cerebrospinal fluid.
63 . The method of claim 61 , wherein the biological sample comprises at least one component selected from the group consisting of a throat swab, nasal swab, vaginal swab, and rectal swab.
64 . The method of claim 61 , wherein the biological sample comprises a biopsy sample.
65 . The method of claim 55 , wherein the crude matrix is not subjected to a lysis treatment.
66 . The method of claim 55 , wherein the crude matrix is not treated with a chaotropic agent, a detergent, or a lytic enzyme preparation.
67 . The method of claim 55 , wherein the crude matrix is not subjected to a high temperature thermal treatment.
68 . The method of claim 55 , wherein the target nucleic acid species is a Staphylococcus spp. nucleic acid.
69 . The method of claim 68 , wherein the Staphylococcus spp. nucleic acid is from S. aureus.
70 . The method of claim 69 , wherein the S. aureus is methicillin-resistant S. aureus (MRSA).
71 . The method of claim 55 , wherein the target nucleic acid species is a mycoplasma nucleic acid.
72 . The method of claim 55 , wherein the crude matrix is subjected to a lysis treatment.
73 . The method of claim 72 , wherein the lysis treatment comprises treating the crude matrix with a detergent.
74 . The method of claim 72 , wherein the lysis treatment comprises treating the crude matrix with a lytic enzyme.
75 . The method of claim 74 , wherein the lytic enzyme is PlyC.
76 . The method of claim 55 , wherein the target nucleic acid species is a Streptococcus spp. nucleic acid.
77 . The method of claim 55 , wherein the Streptococcus spp. nucleic acid is from a group A Streptococcus spp. (Strep A).
78 . The method of claim 55 , wherein the target nucleic acid species is a Salmonella spp. nucleic acid.
79 . The method of claim 78 , wherein the Salmonella spp. nucleic acid is from S. typhimurium.
80 . The method of claim 55 , wherein the target nucleic acid is a bacterial nucleic acid.
81 . The method of claim 80 , wherein the bacteria nucleic acid is from the group consisting of Chlamydia trachomatis, Neisseria gonorrhea , a Group A Streptococcus spp., a Group B Streptococcus spp., Clostridium difficile, Escherichia coli, Mycobacterium tuberculosis, Helicobacter pylori, Gardnerella vaginalis, Mycoplasma hominis , a Mobiluncus spp., a Prevotella spp., and a Porphyromonas spp.
82 . The method of claim 55 , wherein the target nucleic acid is a mammalian nucleic acid.
83 . The method of claim 82 , wherein the target nucleic acid is associated with tumor cells.
84 . The method of claim 55 , wherein the target nucleic acid is a viral nucleic acid.
85 . The method of claim 84 , wherein the viral nucleic acid is from human immunodeficiency virus, influenza virus, or dengue virus.
86 . The method of claim 55 , wherein the target nucleic acid is a fungal nucleic acid.
87 . The method of claim 86 , wherein the fungal nucleic acid is from Candida albicans.
88 . The method of claim 55 , wherein the target nucleic acid is a protozoan nucleic acid.
89 . The method of claim 88 , wherein the protozoan nucleic acid is from a Trichomonas spp.
90 . The method of claim 55 , wherein the isothermal nucleic acid amplification reaction is a recombinase polymerase amplification reaction.
91 . The method of claim 55 , wherein the isothermal nucleic acid amplification reaction is selected from the group consisting of transcription-mediated amplification, nucleic acid sequence-based amplification, signal mediated-amplification of RNA, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, circular helicase-dependent amplification, and nicking and extension amplification reaction.
92 . The method of claim 55 , wherein the mixture comprises polyethylene glycol (PEG).
93 . The method of claim 92 , wherein PEG is present in the mixture at a concentration of greater than 1%.
94 . A method for detection of a target nucleic acid, the method comprising:
contacting a sample comprising a target nucleic acid with a reaction rehydration buffer or a hydrated reaction system; and
amplifying the target nucleic acid in the sample to a detectable level,
wherein the sample is not treated with a chaotropic agent, a detergent, a lytic enzyme preparation, or subjected to a high temperature thermal treatment prior to contacting the sample with the reaction hydration buffer or the hydrated reaction system.
95 . The method of claim 94 , wherein the target nucleic acid comprises genomic DNA of Staphylococcus aureus.
96 . The method of claim 95 , wherein the target nucleic acid comprises genomic DNA of methicillin-resistant Staphylococcus aureus.
97 . The method of claim 94 , wherein the amplification is performed using recombinase polymerase amplification.
98 . The method of claim 94 , wherein the rehydration buffer or the rehydrated reaction system comprises polyethylene glycol at a concentration of greater than 1%.
99 . A kit comprising:
components of an isothermal nucleic acid amplification reaction; and
a lateral flow device, a microfluidic device, or a swab.
100 . The kit of claim 99 , wherein the kit does not comprise reagents for nucleic acid purification or extraction.