METHOD, SYSTEM AND APPARATUS FOR DETECTION
The use of Nucleic Acid Amplification Technologies (NAATs) to rapidly copy a specific fragment of DNA from a few starting molecules has been used to determine the presence of that DNA in a sample. It is of importance for various applications including the identification of a pathogen in a clinical sample. The disclosed embodiments describe an apparatus, disc, methods, and a system for detecting microorganisms such as pathogenic viruses and bacterial rapidly.
1 . A method of detecting a nucleic acid of one or more microorganism in a subject, the method comprising, independent of order, the following steps:
a. obtaining an upper respiratory sample from a subject;
b. processing the upper respiratory sample in an apparatus to capture and lyse microorganisms from the sample, and obtaining a nucleic acid extract from microorganisms in the upper respiratory sample of a subject;
c. selecting one or more target sequence from a microorganism of interest, and selecting one or more nucleic acid amplification primer set that is complementary to at least a portion of a target sequence from a microorganism of interest;
d. incubating the target sequence with the one or more nucleic acid amplification primer set in a reaction mixture and performing an amplification reaction; and
e. detecting one or more target sequence from a microorganism of interest.
2 . A method according to embodiment 1, wherein the incubation step includes a pre-amplification step before step d) that uses random primers and reagents for the nonselective amplification of nucleic acid from microorganisms in the sample to produce a pre-amplification product.
3 . A method according to embodiment 1, wherein an upper respiratory sample from a subject comprised samples from a nasal pharyngeal swab, a nasal swab, a throat swab, saliva, a nasal aspirate, and any other method suitable to obtain sufficient sample.
4 . A method according to embodiment 1, wherein more than one microorganism in a subject's upper respiratory sample can be detected.
5 . A method according to embodiment 1, wherein the microorganism comprises a virus.
6 . A method according to embodiment 4, wherein the virus comprises a coronavirus
7 . A method according to embodiment 4, wherein the virus is a SARS-CoV-2 type virus.
8 . A method according to embodiment 4, wherein the virus is selected from Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4Respiratory Syncytial Virus, and SARS-CoV-2 type virus.
9 . A method according to embodiment 1, wherein the amplified template is detected or quantified in real time.
10 . A method according to embodiment 1, wherein the amplification is isothermal.
11 . A method according to embodiment 1, wherein the target sequence comprises a SARS-CoV-2 type virus nucleic acid sequence in the nucleocapsid recombinant N2 fragment domain or in the nucleocapsid recombinant N3 fragment domain.
12 . A method according to embodiment 1, wherein the target sequence comprises a SARS-CoV-2 type virus nucleic acid sequence in the nucleocapsid recombinant N2 fragment domain the target sequence comprises a nucleocapsid recombinant N3 fragment domain.
13 . A method according to embodiment 1, wherein the target sequence comprises a SARS-CoV-2 type virus nucleic acid sequence and the primers that are complementary to at least a portion of that target sequence are selected from CTGAGGGAGCCTTGAATACACCAA (SEQ ID NO:1); CGCCATTGCCAGCCATTCTAGC (SEQ ID NO:2); TCCCTTCTGCGTAGAAGCCTTTTGGC-CCCGCAATCCTGCTAACAATGCT (SEQ ID NO:3); CAGAGGCGGCAGTCAAGCCTCTTC-CCCCTACTGCTGCCTGGAGTT (SEQ ID NO:4); GTTGTTCCTTGAGGAAGTTGTAGCACGA (SEQ ID NO:5); CGTTCCTCATCACGTAGTCGCAACAG (SEQ ID NO:6); ATGGAGAACGCAGTGGGGC (SEQ ID NO:7); TCATTTTACCGTCACCACCACGAA (SEQ ID NO:8); GCCATGTTGAGTGAGAGCGGTGAACC-GCGATCAAAACAACGTCGGCC (SEQ ID NO:9); AATTCCCTCGAGGACAAGGCGTTCCA-TGGTAGCTCTTCGGTAGTAGCCAA (SEQ ID NO:10); AGACGCAGTATTATTGGGTAAACCTTGG (SEQ ID NO:11); and ATTAACACCAATAGCAGTCCAGATGACCA (SEQ ID NO:12).
14 . A method according to embodiment 1, wherein the target sequence comprises a C. auris nucleic acid sequence and the primers that are complementary to at least a portion of that target sequence are selected from CGGCGAGTTGTAGTCTGGA (SEQ ID NO:13); TCCATCACTGTACTTGTTCGCT (SEQ ID NO:14); GGGCCACAGGAAGCACTAGCACAGCAGGCAAGTCCTTTGG (SEQ ID NO:15); CCGACGAGTCGAGTTGTTTGGGCGGTCTCTCGCCAATATTTAGC (SEQ ID NO:16); AAAGCAGGTACGGGGCTG (SEQ ID NO:17); and GCAGCTCTAAGTGGGTGGTA (SEQ ID NO:18).
15 . A kit for detecting or quantifying a target nucleic acid in a nucleic acid sample, the kit comprising a solid phase disc for detecting nucleic acids comprising one or more amplification primer sets and one or more second primer sets; and ii) instructions for use of the disk for a method of detecting a microorganism in a nucleic acid sample from a subject on an apparatus, instrument, or system described herein.
16 . A method of detecting a nucleic acid of one or more microorganism in a subject, the method comprising, independent of order, the following steps:
a) obtaining a blood or blood fraction sample from a subject;
b) processing the blood sample in an apparatus to capture and lyse microorganisms from the sample, and obtaining a nucleic acid extract from microorganisms in the blood sample of a subject;
c) selecting one or more target sequence from a microorganism of interest, and selecting one or more nucleic acid amplification primer set that is complementary to at least a portion of a target sequence from a microorganism of interest;
d) incubating the target sequence with the one or more nucleic acid amplification primer set in a reaction mixture and performing an amplification reaction; and
e) detecting one or more target sequence from a microorganism of interest.
17 . A method according to embodiment 17, wherein the incubation step includes a pre-amplification step before step d) that uses random primers and reagents for the nonselective amplification of nucleic acid from microorganisms in the sample to produce a pre-amplification product.
18 . A method according to embodiment 17, wherein more than one microorganism in a subject's blood sample can be detected.
19 . A method according to embodiment 17, wherein the microorganism comprises one or more bacteria species.
20 . A method according to embodiment 20, wherein the one or more bacteria species is selected from Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Escherichia Coli, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella, Proteus mirabilis, Citrobacter freundii, Serratia marcescens, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis , and Streptococcus pneumoniae.