Nicking and extension amplification reaction (near) of streptococcus species

Abstract

This invention relates to compositions, methods and kits for detecting the presence or absence of a bacterial species in a biological sample using isothermal nucleic acid amplification.

1 .- 47 . (canceled) 
     
     
         48 . A composition comprising:
 a forward template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the  Streptococcus pyogenes  ( S. pyogenes  ) cell envelope proteinase A (cepA) gene antisense strand; and   a reverse template comprising a nucleotide sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the  S. pyogenes  cepA gene sense strand,   wherein the forward template recognition region is 8-30 nucleotides in length and the reverse template recognition region is 8-30 nucleotides in length.   
     
     
         49 . The composition of  claim 48 , wherein the forward template further comprises a nicking enzyme binding site and a nicking site upstream of the forward template recognition region; and comprises a stabilizing region upstream of the nicking site. 
     
     
         50 . The composition of  claim 48 , wherein the reverse template further comprises a nicking enzyme binding site and a nicking site upstream the reverse template recognition region; and comprises a stabilizing region upstream of the nicking site. 
     
     
         51 . The composition of  claim 48 , further comprising a probe oligonucleotide comprising a sequence complementary to the  S. pyogenes  cepA gene nucleotide sequence. 
     
     
         52 . The composition of  claim 48 , further comprising a DNA polymerase; a nicking enzyme; and dNTPs or a mixture of dNTPs and ddNTPs. 
     
     
         53 . The composition of  claim 48 , wherein the composition is lyophilized. 
     
     
         54 . The composition of  claim 48 , wherein the forward template and/or the reverse template comprises a modified nucleotide, a spacer, or a blocking group. 
     
     
         55 . The composition of  claim 48 , wherein the forward template and/or the reverse template comprises a modified nucleotide comprising a 2′ modification. 
     
     
         56 . The composition of  claim 48 , wherein the forward template and/or the reverse template comprises a modified nucleotide comprising a 2′-O-methyl. 
     
     
         57 . The composition of  claim 48 , wherein the forward template and/or the reverse template comprises a phosphorothioate.