Primers for isothermal amplification of hepatitis C virus

Abstract

The present application relates to primers for isothermal amplification of HCV each include at least eighteen consecutive bases corresponding to a 3′ end region of one selected from base sequences of SEQ ID NOs: 1-10, 21 and 22. The primers are specific to HCV subtypes 1 a, 1 b, 2 a, 2 b and 3 a , respectively and enable genotyping of HCV by isothermal amplification.

1 . A primer set for isothermal amplification of hepatitis C virus containing at least one pair of primers each of which comprises at least eighteen consecutive bases corresponding to a 3′ end region of one base sequence selected from the group consisting of SEQ ID NOs: 1-10, 21 and 22.  
     
     
         2 . The primer set according to  claim 1 , wherein at least one of the pair of primers further comprises a T7 promoter sequence.  
     
     
         3 . The primer set according to  claim 1  containing at least one pair of primes selected from the following (1) to (5): 
 (1) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 1 and 2, respectively;    (2) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 3 and 4, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 3 and 9, respectively;    (3) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 5 and 6, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 5 and 10, respectively;    (4) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 7 and 8, respectively; and    (5) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 21 and 22, respectively.    
     
     
         4 . The primer set according to  claim 3 , wherein at least one of the pair of primers further comprises a T7 promoter sequence.  
     
     
         5 . A method for detecting hepatitis C virus, comprising the steps of: 
 subjecting a clinical sample to isothermal amplification with at least one pair of primers selected from the following pairs of primers (1) to (5); and    determining a subtype of hepatitis C virus based on the resulting amplified product:    (1) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 1 and 2, respectively;    (2) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 3 and 4, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 3 and 9, respectively;    (3) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 5 and 6, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 5 and 10, respectively;    (4) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 7 and 8, respectively; and    (5) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 21 and 22, respectively.    
     
     
         6 . The method according to  claim 5 , wherein at least one of the pair of primers further comprises a T7 promoter sequence.  
     
     
         7 . The method according to  claim 5 , further comprising simultaneously determining two or more subtypes of hepatitis C virus with two or more pairs of primers.  
     
     
         8 . A kit for detecting hepatitis C virus, comprising at least one pair of primers selected from the following pairs of primers (1) to (5); and 
 at least one probe containing a sequence complementary to an amplified product amplified by the action of the at least one pair of primers and serving to detect the amplified product:    (1) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 1 and 2, respectively;    (2) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 3 and 4, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 3 and 9, respectively;    (3) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 5 and 6, respectively, or a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 5 and 10, respectively;    (4) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 7 and 8, respectively; and    (5) a pair of primers comprising at least eighteen consecutive bases corresponding to a 3′ end region of the base sequences of SEQ ID NOs: 21 and 22, respectively.    
     
     
         9 . The kit according to  claim 8 , wherein at least one of the at least one pair of primers further comprises a T7 promoter sequence.