Methods for rapid detection and identification of viral nucleic acids

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.

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.

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.

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.

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.

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.

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.