Method for mutation detection in HIV-1 using pol sequencing

Abstract

The present invention relates to a method for mutation analysis of the HIV pol gene of HIV-1 virions comprising amplifying virion RNA or DNA via nested PCR using outer primers as represented in SEQ ID No. 1 and 2, amplifying said PCR product via nested PCR using a 5′ and 3′ primer chosen from the inner primers SEQ ID No. 3, 4, 5 and 6, and sequencing this secondary obtained PCR product using at least one sequencing primer chosen from any of SEQ ID No. 7 to 12 or variants thereof. In the alternative, at least one secondary sequencing primer may be used chosen from any of SEQ ID No. 13 to 24. The present invention also relates to kits for performing such a method as well as primers for performing the same.

1 . Method for mutation analysis of the pol gene of HIV-1 isolates comprising the steps of: 
 a) isolation of a sample,    b) virion RNA extraction of the isolated sample material,    c) amplifying RNA via nested PCR using outer primers as represented in SEQ ID No. 1 and 2,    d) amplifying said PCR product via nested PCR using a 5′ and 3′ primer chosen from the inner primers as represented in SEQ ID No. 3, 4, 5 and 6, and    e) sequencing this secondary obtained PCR product using at least one sequencing primer chosen from any of SEQ ID No. 7 to 12.    
     
     
         2 . Method for mutation analysis of the pol gene of HIV-1 isolates comprising the steps of: 
 a) isolation of a sample,    b) viral DNA extraction of the isolated sample material,    c) amplifying DNA via nested PCR using outer primers as represented in SEQ ID No. 1 and 2,    d) amplifying said PCR product via nested PCR using a 5′ and 3′ primer chosen from the inner primers as represented in SEQ ID No. 3, 4, 5 and 6, and    e) sequencing this secondary obtained PCR product using at least one sequencing primer chosen from any of SEQ ID No. 7 to 12.    
     
     
         3 . Method according to claims  1  or  2  wherein one of the initial sequencing primers is replaced by one or a pair of replacement primers.  
     
     
         4 . Method according to any of  claims 1  to  3  wherein the sequencing primer is chosen up to 1, 2, 3 or 4 nucleotides upstream or downstream the described primer region.  
     
     
         5 . Method according to any of  claims 1  to  3  wherein the outer primer is chosen up to 1, 2, 3 or 4 nucleotides upstream or downstream the described primer region.  
     
     
         6 . Method according to any of  claims 1  to  3  wherein the inner primer is chosen up to 1, 2, 3 or 4 nucleotides upstream or downstream the described primer region.  
     
     
         7 . Method according to any of  claims 1  to  6  wherein the sample contains free virion particles or virus infected cells.  
     
     
         8 . A primer as represented in SEQ ID No. 1 and 5 to 12 used to analyse the sequence of the HIV pol gene of HIV-1 isolates.  
     
     
         9 . Method according to any of  claims 1  to  6  wherein the sequencing is performed on the primary PCR product.  
     
     
         10 . Diagnostic kit for the mutation analysis of the HIV pol gene of HIV-1 isolates comprising at least one of the primers having the sequence as represented in SEQ ID No. 1 to 12.