Methods for rapid detection and identification of viral nucleic acids

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.

We claim: 
     
         1 . A method of detecting presence of human immunodeficiency virus (HIV) nucleic acid in a sample, comprising:
 contacting the sample with a set of loop-mediated isothermal amplification (LAMP) primers specific for an HIV nucleic acid under conditions sufficient for amplification of the HIV nucleic acid, wherein the set of LAMP primers comprises six primers comprising a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 38-48 and 81, thereby producing an HIV amplification product; and   detecting the HIV amplification product, thereby detecting presence of HIV nucleic acid in the sample.   
     
     
         2 . The method of  claim 1 , wherein the set of LAMP primers comprises six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 38, 41, 42, 45, 47, and 48. 
     
     
         3 . A method of detecting presence of one or more viral nucleic acids comprising one or more of human immunodeficiency virus (HIV) nucleic acid, hepatitis B virus (HBV) nucleic acid, hepatitis C virus (HCV) 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 HIV nucleic acid, an HBV nucleic acid, an HCV nucleic acid, an HEV nucleic acid, a WNV nucleic acid or 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 the one or more viral nucleic acid amplification products.   
     
     
         4 . The method of  claim 2 , wherein the one or more sets of LAMP primers comprise:
 (a) 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;   (b) 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;   (c) 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 32-37;   (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 SEQ ID NOs: 56-61; or   (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, SEQ ID NOs: 66-71, SEQ ID NOs: 68-71, SEQ ID NOs: 72-75, or SEQ ID NOs: 66, 67, and 72-75.   
     
     
         5 . The method of  claim 3  or  claim 4 , 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, SEQ ID NOs: 13 and 15-19, or SEQ ID NOs: 14-19. 
     
     
         6 . The method of  claim 3  or  claim 4 , 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, SEQ ID NOs: 13 and 15-19, or SEQ ID NOs: 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 SEQ ID NOs: 56-61. 
     
     
         7 . The method of  claim 3  or  claim 4 , 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 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. 
     
     
         8 . The method of  claim 3  or  claim 4 , 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 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. 
     
     
         9 . The method of  claim 3  or  claim 4 , 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 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; 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; 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 SEQ ID NOs: 56-61; and 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, SEQ ID NOs: 66-71, SEQ ID NOs: 68-71, SEQ ID NOs: 72-75, or SEQ ID NOs: 66, 67, and 72-75. 
     
     
         10 . The method of any one of  claims 3  to  9 , wherein the sample is contacted with the one or more sets of LAMP primers in a single reaction vessel. 
     
     
         11 . A method of detecting presence of hepatitis B virus (HBV) nucleic acid in a sample, comprising:
 contacting the sample with a set of loop-mediated isothermal amplification (LAMP) primers specific for an HBV nucleic acid under conditions sufficient for amplification of the HBV nucleic acid, wherein the set of LAMP primers comprises primers comprising a nucleic acid sequence having at least 90% sequence identity to each of SEQ ID NOs: 1-6, thereby producing an HBV amplification product; and   detecting the HBV amplification product, thereby detecting presence of HBV nucleic acid in the sample.   
     
     
         12 . The method of  claim 11 , wherein the set of LAMP primers comprises six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-6. 
     
     
         13 . A method of detecting presence of hepatitis C virus (HCV) nucleic acid in a sample, comprising:
 contacting the sample with at least one set of loop-mediated isothermal amplification (LAMP) primers specific for an HCV nucleic acid under conditions sufficient for amplification of the HCV nucleic acid, wherein the set of LAMP primers comprises a set of primers comprising a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 7-37, thereby producing an HCV amplification product; and   detecting the HCV amplification product, thereby detecting presence of HCV nucleic acid in the sample.   
     
     
         14 . The method of  claim 13 , wherein the set of LAMP primers comprises six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 7-12, SEQ ID NOs: 13 and 15-19, or SEQ ID NOs: 14-19. 
     
     
         15 . The method of  claim 14 , wherein the at least one set of LAMP primers is specific for an HCV genotype 1 (HCV-1) nucleic acid, an HCV genotype 2 (HCV-2) nucleic acid, or an HCV genotype 3 (HCV-3) nucleic acid. 
     
     
         16 . The method of  claim 15 , wherein the set of LAMP primers is specific for an HCV-1 nucleic acid and comprises six primers comprising or consisting of SEQ ID NOs: 20-25. 
     
     
         17 . The method of  claim 15 , wherein the set of LAMP primers is specific for an HCV-2 nucleic acid and comprises six primers comprising or consisting of SEQ ID NOs: 26-31. 
     
     
         18 . The method of  claim 15 , wherein the set of LAMP primers is specific for an HCV-3 nucleic acid and comprises six primers comprising or consisting of SEQ ID NOs: 32-37. 
     
     
         19 . A method of detecting presence of hepatitis E virus (HEV) nucleic acid in a sample, comprising:
 contacting the sample with a set of loop-mediated isothermal amplification (LAMP) primers specific for an HEV nucleic acid under conditions sufficient for amplification of the HEV nucleic acid, wherein the set of LAMP primers comprises primers comprising a nucleic acid sequence having at least 90% sequence identity to each of SEQ ID NOs: 49-54, thereby producing an HEV amplification product; and   detecting the HEV amplification product, thereby detecting presence of HEV nucleic acid in the sample.   
     
     
         20 . The method of  claim 19 , wherein the set of LAMP primers comprises six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 49-54. 
     
     
         21 . A method of detecting presence of West Nile virus (WNV) nucleic acid in a sample, comprising:
 contacting the sample with a set of loop-mediated isothermal amplification (LAMP) primers specific for an WNV nucleic acid under conditions sufficient for amplification of the WNV nucleic acid, wherein the set of LAMP primers comprises one or more primers comprising a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 55-61, thereby producing an WNV amplification product; and   detecting the WNV amplification product, thereby detecting presence of WNV nucleic acid in the sample.   
     
     
         22 . The method of  claim 21 , wherein the set of LAMP primers comprises six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 55 and 57-61 or SEQ ID NOs: 56-61. 
     
     
         23 . A method of detecting presence of Dengue virus (DENV) nucleic acid in a sample, comprising:
 contacting the sample with a set of loop-mediated isothermal amplification (LAMP) primers specific for an DENV nucleic acid under conditions sufficient for amplification of the DENV nucleic acid, wherein the set of LAMP primers comprises one or more primers comprising a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 62-75, thereby producing an DENV amplification product; and   detecting the DENV amplification product, thereby detecting presence of DENV nucleic acid in the sample.   
     
     
         24 . The method of  claim 23 , wherein the set of LAMP primers comprises four to six primers comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 62-67, SEQ ID NOs: 66-71, SEQ ID NOs: 68-71, SEQ ID NOs: 72-75, or SEQ ID NOs: 66, 67, and 72-75. 
     
     
         25 . The method of any one of  claims 1  to  24 , wherein at least one primer in the set of LAMP primers comprises a detectable label. 
     
     
         26 . The method of  claim 25 , wherein the detectable label comprises a fluorophore. 
     
     
         27 . The method of  claim 25  or  26 , wherein the primer further comprises a fluorescence quencher. 
     
     
         28 . The method of  claim 27 , wherein the fluorescence quencher comprises a dark quencher. 
     
     
         29 . The method of any one of  claims 25  to  28 , 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. 
     
     
         30 . The method of any one of  claims 1  to  29 , further comprising contacting the sample with a reverse transcriptase under conditions sufficient for reverse transcription of the viral nucleic acid. 
     
     
         31 . The method of any one of  claims 1  to  30 , wherein detecting the viral nucleic acid amplification product comprises turbidity measurement, fluorescence detection, or gel electrophoresis. 
     
     
         32 . The method of any one of  claims 1  to  31 , wherein the sample comprises isolated DNA, isolated RNA, blood, plasma, serum, urine, saliva, tissue biopsy, fine needle aspirate, or a surgical specimen. 
     
     
         33 . An isolated nucleic acid primer comprising a nucleic acid sequence at least 90% identical to any one of SEQ ID NOs: 1-75 and 81. 
     
     
         34 . The isolated nucleic acid primer of  claim 33 , comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-75 and 81. 
     
     
         35 . The isolated nucleic acid primer of  claim 34 , consisting of the nucleic acid sequence of any one of SEQ ID NOs: 1-75 and 81. 
     
     
         36 . The isolated nucleic acid primer of any one of  claims 33  to  35 , further comprising a fluorophore, a fluorescent quencher, or both. 
     
     
         37 . The isolated nucleic acid primer of  claim 36 , wherein the nucleic acid sequence of the primer comprises or consists of SEQ ID NOs: 6, 12, 19, 25, 31, 37, 48, 54, 61, or 67. 
     
     
         38 . A kit comprising at least one set of LAMP primers in a container, wherein the at least one set of LAMP primers comprises:
 (a) 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;   (b) 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;   (c) 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 32-37;   (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 SEQ ID NOs: 56-61; or   (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, SEQ ID NOs: 66-71, SEQ ID NOs: 68-71, SEQ ID NOs: 72-75, or SEQ ID NOs: 66, 67, and 72-75.   
     
     
         39 . The kit of  claim 38 , wherein the kit comprises two or more sets of LAMP primers in a single container. 
     
     
         40 . The kit of  claim 39 , wherein the kit comprises 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, SEQ ID NOs: 13 and 15-19, or SEQ ID NOs: 14-19 in a single container. 
     
     
         41 . The kit of  claim 39 , wherein the kit comprises 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, SEQ ID NOs: 13 and 15-19, or SEQ ID NOs: 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 SEQ ID NOs: 56-61 in a single container. 
     
     
         42 . The kit of  claim 39 , wherein the kit comprises 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 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 in a single container. 
     
     
         43 . The kit of  claim 39 , wherein the kit comprises 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 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 in a single container. 
     
     
         44 . The kit of  claim 39 , wherein the kit comprises 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, SEQ ID NOs: 13 and 15-19, SEQ ID NOs: 14-19, SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, or SEQ ID NOs: 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; 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; 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 SEQ ID NOs: 56-61; and 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, SEQ ID NOs: 66-71, SEQ ID NOs: 68-71, SEQ ID NOs: 72-75, or SEQ ID NOs: 66, 67, and 72-75 in a single container. 
     
     
         45 . The kit of any one of  claims 38  to  44 , further comprising a buffer comprising 2% D-mannitol, 0.2% Triton®-X100, 40 mM Tris-HCl, 20 mM KCl, 20 mM (NH 4 ) 2 SO 4 , 6 mM MgSO 4 , 0.5 M L-proline, 10 mM Tris acetate, 1.6 mM magnesium acetate, 15 mM potassium acetate and 2 mM each of dATP, dCTP, dGTP, and dTTP. 
     
     
         46 . A nucleic acid amplification buffer comprising 2% D-mannitol, 0.2% Triton®-X100, 40 mM Tris-HCl, 20 mM KCl, 20 mM (NH 4 ) 2 SO 4 , 6 mM MgSO 4 , 0.5 M L-proline, 10 mM Tris acetate, 1.6 mM magnesium acetate, 15 mM potassium acetate and 2 mM each of dATP, dCTP, dGTP, and dTTP. 
     
     
         47 . The buffer of  claim 46 , wherein the buffer has a pH of about 7.8.