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 for determining the presence or absence of a pathogen in a stool sample without nucleic acid extraction or purification, comprising;
i) applying a stool sample to an assay device;
ii) lysing the sample within the assay device to release intracellular nucleic acid of the pathogen;
iii) contacting the sample with a recombinase polymerase amplification (RPA) reagent mixture within the assay device, wherein the RPA reagent mixture comprises primers specific for the pathogen nucleic acid, a UvsX recombinase, and a UvsY loading agent;
iv) amplifying the target nucleic acid; and
v) determining the presence or absence of the pathogen nucleic acid based on an increased signal from a nucleic acid probe within the assay device.
56 . The method of claim 55 , wherein the assay device is selected from the group consisting of a microfluidic device, a lateral flow device, and similar such assay device.
57 . The method of claim 55 , wherein the pathogen is selected from the group consisting of the species Acinetobacter, Aerococcus, Bacteroides, Bordetella, Campylobacter, Clostridium, Corynebacterium, Chlamydia, Citrobacter, Enterobacter, Enterococcus, Escherichia, Helicobacter, Haemophilus, Klebsiella, Legionella, Listeria, Micrococcus, Mobilincus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Oligella, Pasteurella, Prevotella, Porphyromonas, Pseudomonas, Propionibacterium, Proteus, Salmonella, Serratia, Staphylococcus, Streptococcus, Treponema, Bacillus, Francisella , and Yersinia.
58 . The method of claim 55 , wherein lysing the sample comprises contacting the sample with a detergent and/or a lytic enzyme.
59 . The method of claim 58 , wherein the detergent comprises Triton-X 100.
60 . The method of claim 58 , wherein the lytic enzyme comprises bacteriophage lysin.
61 . The method of claim 60 , wherein the bacteriophage lysin comprises Streptococcal Ci bacteriophage lysin (PlyC).
62 . The method of claim 55 , wherein the RPA reagent mixture further comprises a gp32 single strand binding protein, a polymerase, and a crowding agent.
63 . The method of claim 55 , wherein the UvsX is a T4 phage UvsX and the UvsY is a T4 phage UvsY.
64 . The method of claim 62 , wherein the gp32 is a T4 phage gp32, the polymerase is a Bacillus subtilis polymerase I large fragment, and the crowding agent is polyethylene glycol (PEG).
65 . The method of claim 55 , wherein the RPA reagent mixture is provided in dried form within the assay device.
66 . The method of claim 65 , wherein the dried RPA reagent mixture is provided as freeze-dried or lyophilized powder, pellet or bead.
67 . The method of claim 55 , wherein the nucleic acid probe is a fluorescent probe.
68 . The method of claim 67 , wherein the fluorescent probe is selected from the group consisting of fluorescein, TAMRA, and Fam.