# US 11,306,367 B1 · Methods for rapid detection and identification of viral nucleic acids

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- Number: US 11,306,367 B1
- Document ID: US11306367B1
- Status: Granted
- Kind: B1
- Application number: 14/821,865
- Filed: 2015-08-10
- Granted: 2022-04-19
- Earliest claimed: 2014-04-14
- Applicants: Dougbeh-Chris Nyan
- Inventors: Dougbeh-Chris Nyan
- Web page: https://patentgrep.com/patent/US11306367B1

## CPC

- C12Q 1/703: {Viruses associated with AIDS}
- C12Q 1/701: {Specific hybridization probes}
- C12Q 1/706: {for hepatitis}
- C12Q 1/707: {non-A, non-B Hepatitis, excluding hepatitis D}
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Disclosed herein are methods of detecting viral nucleic acids in a sample that include contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for a viral nucleic acid of interest (such as hepatitis B virus, hepatitis C virus, hepatitis E virus, human immunodeficiency virus, West Nile virus, or Dengue virus nucleic acids) under conditions sufficient to produce an amplification product and detecting the amplification product(s). In some examples, the amplification product is detected by gel electrophoresis, while in other examples, the amplification product is detected by detecting signal from a label included in one or more of the LAMP primers. Primers and kits for use for detection of viral nucleic acids by LAMP are also disclosed herein.

## Claims

### Claim 1

A method of detecting a presence of viral nucleic acids comprising hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, and Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids if present in the sample, thereby producing one to a plurality of viral nucleic acid amplification products depending on the number of viral nucleic acids in the sample; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the one or more sets of LAMP primers are selected from primers comprising a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-75 and 81;
wherein at least one primer in the set of LAMP primers comprises a detectable label;
wherein all SEQ ID NO: 1-75 and 81 are present in the method;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns.

### Claim 2

A method of detecting a presence of viral nucleic acids comprising hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, and Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids if present in the sample, thereby producing one to a plurality of viral nucleic acid amplification products depending on the number of viral nucleic acids in the sample; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the one or more sets of LAMP primers comprise:
(a) a set of LAMP primers specific for an HBV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6;
(b) a set of LAMP primers specific for an HCV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 7-12, 13 and 15-19, 14-19, 20-25, 26-31, or 32-37;
(c) a set of LAMP primers specific for an HIV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 38, 41, 42, 45, 47, and 48;
(d) a set of LAMP primers specific for an HEV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 49-54;
(e) a set of LAMP primers specific for a WNV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 55 and 57-61 or 56-61; and
(f) a set of LAMP primers specific for a DENV nucleic acid comprising four to six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 62-67, 66-71, 68-71, 72-75, or 66, 67, and 72-75;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns.

### Claim 3

A method of detecting presence of viral nucleic acids comprising one or more of hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, or Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for one or more of an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids, thereby producing one or more viral nucleic acid amplification products; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the method comprises contacting the sample with a set of LAMP primers specific for an HBV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6 and a set of LAMP primers specific for an HCV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 7-12, 13 and 15-19, or 14-19;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns.

### Claim 4

A method of detecting presence of viral nucleic acids comprising one or more of hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, or Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for one or more of an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids, thereby producing one or more viral nucleic acid amplification products; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the method comprises contacting the sample with a set of LAMP primers specific for an HBV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6, a set of LAMP primers specific for an HCV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 7-12, 13 and 15-19, or 14-19, and a set of LAMP primers specific for a WNV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 55 and 57-61 or 56-61;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns.

### Claim 5

A method of detecting presence of viral nucleic acids comprising one or more of hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, or Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for one or more of an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids, thereby producing one or more viral nucleic acid amplification products; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the method comprises contacting the sample with a set of LAMP primers specific for an HBV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6; a set of LAMP primers specific for an HCV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 7-12, 13 and 15-19, 14-19, 20-25, 26-31, or 32-37; and a set of LAMP primers specific for an HIV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 38, 41, 42, 45, 47, and 48;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns.

### Claim 6

A method of detecting presence of viral nucleic acids comprising one or more of hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, or Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for one or more of an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids, thereby producing one or more viral nucleic acid amplification products; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the method comprises contacting the sample with a set of LAMP primers specific for an HBV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6; a set of LAMP primers specific for an HCV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 7-12, 13 and 15-19, 14-19, 20-25, 26-31, or 32-37; a set of LAMP primers specific for an HIV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 38, 41, 42, 45, 47, and 48; and a set of LAMP primers specific for an HEV nucleic acid comprising six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 49-54;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns.

### Claim 7

A method of detecting a presence of viral nucleic acids comprising hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) nucleic acid, human immunodeficiency virus (HIV) nucleic acid, hepatitis E virus (HEV) nucleic acid, West Nile virus (WNV) nucleic acid, and Dengue virus (DENV) nucleic acid in a sample, comprising:
contacting the sample with one or more sets of loop-mediated isothermal amplification (LAMP) primers specific for an HBV nucleic acid, an HCV nucleic acid, an HIV nucleic acid, an HEV nucleic acid, a WNV nucleic acid and a DENV nucleic acid under conditions sufficient for amplification of the one or more viral nucleic acids if present in the sample, thereby producing one to a plurality of viral nucleic acid amplification products depending on the number of viral nucleic acids in the sample; and
detecting any present viral nucleic acid amplification products in the sample;
wherein the sample is contacted with the one or more sets of LAMP primers in a single reaction vessel;
wherein at least one primer in the set of LAMP primers comprises a detectable label;
providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends;
wherein the step of providing a single pathogen specific detector and labelling the single pathogen specific detector at both ends comprising the steps of (i) providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen; (ii) covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide; and (iii) using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns;
wherein all SEQ ID NO: 1-75 and 81 are present in the method.

### Claim 8 (depends on 1)

The method of claim 1 wherein the detectable label comprises a fluorophore.

### Claim 9 (depends on 1)

The method of claim 1 wherein the primer further comprises a fluorescence quencher.

### Claim 10 (depends on 9)

The method of claim 9 wherein the fluorescence quencher comprises a dark quencher.

### Claim 11 (depends on 1)

The method of claim 1 further comprising contacting the sample with a reverse transcriptase under conditions sufficient for reverse transcription of the viral nucleic acid.

### Claim 12

The method of 2 wherein detecting the viral nucleic acid amplification product comprises turbidity measurement, fluorescence detection, or gel electrophoresis.

### Claim 13 (depends on 1)

The method of claim 1 wherein the sample comprises isolated DNA, isolated RNA, blood, plasma, serum, urine, saliva, tissue biopsy, fine needle aspirate, or a surgical specimen.

### Claim 14 (depends on 7)

The method of claim 7 wherein the primer further comprises a dark quencher.

### Claim 15 (depends on 7)

The method of claim 7 wherein the at least one primer comprising the detectable label comprises any one of SEQ ID NOs: 5, 6, 11, 12, 18, 19, 24, 25, 30, 31, 36, 37, 46-48, 53, 54, 60, 61, 66, or 67.

### Claim 16 (depends on 7)

The method of claim 7 further comprising contacting the sample with a reverse transcriptase under conditions sufficient for reverse transcription of the viral nucleic acid.

### Claim 17

The method of 9 wherein detecting the viral nucleic acid amplification product comprises turbidity measurement, fluorescence detection, or gel electrophoresis.

### Claim 18 (depends on 7)

The method of claim 7 wherein the sample comprises isolated DNA, isolated RNA, blood, plasma, serum, urine, saliva, tissue biopsy, fine needle aspirate, or a surgical specimen.

### Claim 19

A one-step single-tube multiplex-reaction for detection of viruses and pathogens infecting human, plants, and animals using a single pathogen specific detector that is labeled at both ends; comprising:
providing a single pathogen-specific Loop Reverse oligonucleotide probe (LRp) per pathogen;
covalently labeling the LRp probe with a fluorophore at a 5′-end and with a quencher at a 3′-end to create a bi-labeled LRp olingonucleotide;
using the bi-labeled LRp oligonucleotide along with a plurality of other primers as a set per pathogen for a one-step single-tube multiplex-reaction;
wherein both fluorescence and quenching are accomplished in a single-tube one-step reaction for simultaneous detection and identification of multiple pathogens with differentiating ladder-like banding patterns;
wherein all SEQ ID NO: 1-75 and 81 are present in the method.