Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification

Abstract

The disclosure relates to novel probes for use in LAMP detection methods. The probes contain a single fluorophore label bound to an internal cytosine residue of the probe. The probes are particularly useful in the detection of chlamydia and gonorrhea infections in a patient.

1 . A probe for isothermal nucleic acid amplification comprising an oligonucleotide probe sequence complementary to a region of a target nucleic acid sequence, wherein said oligonucleotide probe sequence has only one fluorophore label and which label is bound to an internal cytosine base and wherein said oligonucleotide probe sequence does not have a 3′ end terminator. 
     
     
         2 . The probe of  claim 1 , wherein the cytosine base is substantially centrally disposed along the oligonucleotide's length except for positions 1-3 at the 3′ end and position 1 at the 5′ end. 
     
     
         3 . The probe of  claim 1 , wherein the oligonucleotide probe sequence is a DNA sequence and the target nucleic acid sequence is a DNA sequence. 
     
     
         4 . The probe of  claim 1 , wherein the fluorophore comprises any one or more selected from the group consisting of: FAM, JOE, TET, HEX, TAMRA, ROX, ALEXA and ATTO. 
     
     
         5 . The probe of  claim 1 , comprising the following sequence:
 5′ Xn C*Xm 3′   wherein n is >1, m>3, X is nucleotide base; and * is fluorophore.   
     
     
         6 . The probe of  claim 5 , wherein the nucleotide base is selected from the group consisting of A, T, C and G, n is more than 1 to 20 or less and m is more than 3 to 20 or less. 
     
     
         7 . The probe of  claim 1 , comprising one or more of the following sequences:
 SEQ ID NO: 2: TAAGATAAC[C-FAM]CCGCACGTG (CT PB1-FAM internal),   SEQ ID NO: 4: GCGAACATA [C-ALEXA546] CAGCTATGATCAA (GC porA7-joe loopF), or   SEQ ID NO: 5: ATGTTCA [C-JOE] CATGGCGGAG (GC glnA7-ALEXA546 loopB).   
     
     
         8 . The probe of  claim 1 , wherein the target nucleic acid is from a micro-organism, fungi, yeast or virus. 
     
     
         9 . The probe of  claim 1 , wherein the probe is configured to be used in loop-mediated isothermal nucleic acid amplification. 
     
     
         10 . A method of detecting a target nucleic acid sequence in a sample, the method comprising:
 amplifying a target nucleic acid in the sample to provide an amplified nucleic acid;   probing the amplified nucleic acid with a probe as claimed in  claim 1 ; and   detecting the presence of the target nucleic acid, wherein an increases in fluorescence of the probe indicates the presence of the target nucleic acid in the sample.   
     
     
         11 . The method of  claim 10 , wherein the target nucleic acid is from a micro-organism, fungi, yeast or virus. 
     
     
         12 . The method of  claim 10 , wherein the target nucleic acid is from  Chlamydia trachomatis  or  Neisseria gonorrhoeae.    
     
     
         13 . A method of diagnosing  Chlamydia  and/or Gonorrhea infection in a patient, the method comprising
 providing a sample derived from the patient;   adding one or more probes of  claim 1  to the sample; and   detecting the presence of a nucleic acid derived from  Chlamydia trachomatis  and/or  Neisseria gonorrhoeae , wherein an increase in the fluorescence of the probe indicates the presence of a  Chlamydia trachomatis  and/or  Neisseria gonorrhoeae  infection.   
     
     
         14 . The method of  claim 13 , wherein a single type of probe specific for a nucleic acid from either  Chlamydia trachomatis  or  Neisseria gonorrhoeae  is added to the sample. 
     
     
         15 . The method of  claim 13 , wherein at least two different probes are added to the sample wherein a first probe is labelled with a first fluorescent label and is specific for probing  Chlamydia trachomatis  nucleic acid and a second probe is labelled with a different fluorescent label to the first probe and is specific for probing  Neisseria gonorrhoeae  nucleic acid. 
     
     
         16 . The method of  claim 10 , wherein the probes are provided in a buffer system comprising dNTPs at a concentration of from 1-10 mM, one or more salts at a concentration of each salt of from 2-20 mM, Tris pH8.8 at a concentration of from 10-100 mM, Trehalose at a concentration of from 10-100 mM, BST polymerase at an amount of from 1 U-12 U and 0.01%-1% 1,2 propanediol. 
     
     
         17 . The method of  claim 16 , wherein the one or more salts are selected from the group consisting of KCl, (NH 4 ) 2 SO 4  and MgSO 4 . 
     
     
         18 . A kit for detecting a target nucleic acid comprising a probe as claimed in  claim 1 , a loop-mediated isothermal amplification reagent a buffer, an enzyme, dNTPs and one or more loop-mediated isothermal amplification primers. 
     
     
         19 . The kit of  claim 18 , further comprising a positive and negative control. 
     
     
         20 . The kit of  claim 18 , wherein the reagent buffer comprises dNTPs at a concentration of from 1-10 mM, one or more salts at a concentration of from 2-20 mM, Tris pH8.8 at a concentration of from 10-100 mM, Trehalose at a concentration of from 10-100 mM, BST polymerase at an amount of from 1 U-12 U and 0.01%-1% 1,2 propanediol. 
     
     
         21 . The kit of  claim 20 , wherein the one or more salts are selected from the group consisting of KCl, (NH 4 ) 2 SO 4  and MgSO 4 . 
     
     
         22 . The probe of  claim 4 , wherein the fluorophore is FAM, Joe or Alexa546.