Methos 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.

What is claimed is: 
     
         1 . A method of detecting presence of one or more 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 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.   
     
     
         2 . The method of  claim 1 , 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. 
     
     
         3 . The method of  claim 1 , 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; 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, 66-71, 68-71, 72-75, or 66, 67, and 72-75.   
     
     
         4 . The method of  claim 1 , 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. 
     
     
         5 . The method of  claim 1 , 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. 
     
     
         6 . The method of  claim 1 , 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. 
     
     
         7 . The method of  claim 1 , 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. 
     
     
         8 . The method of  claim 1 , 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; 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 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, 66-71, 68-71, 72-75, or 66, 67, and 72-75. 
     
     
         9 . The method of  claim 1 , wherein the sample is contacted with the one or more sets of LAMP primers in a single reaction vessel. 
     
     
         10 . A kit comprising at least one set of LAMP primers in a container. 
     
     
         11 . The kit of  claim 10 , wherein the at least one set of LAMP primers comprises:
 (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; 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, 66-71, 68-71, 72-75, or 66, 67, and 72-75.   
     
     
         12 . The kit of  claim 11 , wherein the kit comprises two or more sets of LAMP primers in a single container. 
     
     
         13 . The kit of  claim 12 , 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, 13 and 15-19, or 14-19 in a single container. 
     
     
         14 . The kit of  claim 12 , 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, 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 in a single container. 
     
     
         15 . The kit of  claim 12 , 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, 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 in a single container. 
     
     
         16 . The kit of  claim 12 , 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, 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 in a single container. 
     
     
         17 . The kit of  claim 12 , 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, 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; 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 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, 66-71, 68-71, 72-75, or 66, 67, and 72-75 in a single container. 
     
     
         18 . The kit of any one of the  claim 10 , 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. 
     
     
         19 . 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. 
     
     
         20 . The buffer of  claim 19 , wherein the buffer has a pH of about 7.8.
 In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.