Solid phase recombinase polymerase amplification for human pathogen identification
The present invention is directed to methods and devices for identification of pathogens, and more particularly to solid phase recombinase polymerase amplification (SP-RPA) methods and devices for massively multiplexed solid-phase isothermal identification of human pathogens from patient samples. The principal application for multiplexed solid-phase isothermal identification of human pathogens is point-of-care diagnostics.
1 . A sample analysis device, comprising:
(a) a sample and reagent input module;
(b) a multiplex reaction module in communication with the input module, the multiplex reaction module comprising a plurality of sample handling chambers for performing recombinase polymerase amplification assay, wherein the recombinase polymerase amplification assay comprises at least one forward primer immobilized on a solid support and at least one reverse primer comprising a detectable label; wherein the forward primer and the reverse primer are complementary to a target nucleic acid;
wherein the at least one forward primer is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11; and
wherein the at least one reverse primer is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12:
(c) a data analysis module in communication with the multiplex reaction module, wherein the data analysis module analyzes for the presence of the detectable label; and
(d) a waste output module for receiving waste from the data analysis module.
2 . The sample analysis device of claim 1 , wherein the plurality of sample handling chambers in the multiplex reaction module comprise 2-500 reaction wells or spots per square millimeter.
3 . The sample analysis device of claim 2 , wherein the plurality of sample handling chambers in the multiplex reaction module comprise approximately 400 reaction wells or spots per square millimeter.
4 . The sample analysis device of claim 1 , wherein the plurality of sample handling chambers in the multiplex reaction module comprise approximately 30,000 wells or spots per microscope slide.
5 . The sample analysis device of claim 1 , wherein the plurality of sample handling chambers in the multiplex reaction module are arranged in a two-dimensional printed array.
6 . The sample analysis device of claim 1 , wherein said target nucleic acid comprises a nucleic acid or nucleotide sequence from a pathogen.
7 . The sample analysis device of claim 6 , wherein said pathogen is a virus, parasite, bacteria, fungi, and combinations thereof.
8 . The sample analysis device of claim 7 , wherein the pathogen is selected from single stranded RNA viruses, single stranded DNA viruses, Zika virus, HIV, hepatitis A, B, and C virus, HSV, CMV EBV, HPV, Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species, mycobacteria, Salmonella , Streptococci, E. coli , Staphylococci, Candida, Aspergillus, Pneumocystis carinii , prions, and combinations thereof.
9 . The sample analysis device of claim 1 , wherein said target nucleic acid is selected from ribosomal RNAs or DNAs, DNA or RNA sequences that are associated with a genetic condition; DNA or RNA sequences for identifying a person; DNA or RNA sequences containing single nucleotide polymorphisms, and combinations thereof.
10 . The sample analysis device of claim 1 , wherein said sample handling chambers each have a volume ranging from 1 μL to 500 μL.
11 . The sample analysis device of claim 1 , wherein said multiplex reaction module comprises a glass substrate that is functionalized with epoxy silane.
12 . The sample analysis device of claim 1 , wherein said detectable label is selected from a fluorescent label, a radioactive label, a chemiluminescence label, an electron spin resonance label, and combinations thereof.
13 . The sample analysis device of claim 1 , wherein said data analysis modules detects radioactive labels, fluorescent labels, chemiluminescent labels, electron spin labels, and combinations thereof.
14 . The sample analysis device of claim 1 , wherein said forward primer and said reverse primer are each complementary to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin, 18S rRNA, ubiquitin, antibiotic resistance genes, toxin genes, CRISPR genes, SNPs, and combinations thereof.
15 . A method of detecting a target nucleic acid in a patient sample, the method comprising the steps of:
(a) obtaining a sample from a patient, the sample containing nucleic acid;
(b) amplifying the nucleic acid on a multiplex array using a recombinase polymerase amplification (RPA) assay with at least one forward primer and at least one reverse primer, wherein the forward primer is immobilized on a solid support and the reverse primer contains a detectable label; and wherein the forward primer and the reverse primer are complementary to the target nucleic acid;
wherein the at least one forward primer is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11; and
wherein the at least one reverse primer is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12; and
(c) detecting for the presence of the detectable label on the target nucleic acid.
16 . The method of claim 15 , wherein the sample is selected from blood, serum, urine, saliva, tissues, cells, and organs, purified or partially purified nucleic acids, or portions thereof.
17 . The method of claim 15 wherein the nucleic acid is a nucleic acid from a pathogen.
18 . The method of claim 17 , wherein the pathogen is a virus, parasite, bacteria, fungi, and combinations thereof.
19 . The method of claim 17 wherein the pathogen is selected from single stranded RNA viruses, single stranded DNA viruses, Zika virus, HIV, hepatitis A, B, and C virus, HSV, CMV EBV, HPV, Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species, mycobacteria, Salmonella , Streptococci, E. coli , Staphylococci, Candida, Aspergillus, Pneumocystis carinii , prions, and combinations thereof.
20 . The method of claim 15 , wherein said forward primer and said reverse primer are each complementary to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin, 18S rRNA, ubiquitin, antibiotic resistance genes, toxin genes, CRISPR genes, SNPs, and combinations thereof.