Methods for multiplex detection of alleles associated with ophthalmic conditions

Abstract

Systems and methods for detecting at least two genomic alleles associated with corneal dystrophy in a sample from a human subject are disclosed in which cells (e.g., epithelial) of the subject are adhered to a tip of a substrate. The tip of the substrate is agitated in a lysis solution that lyses cells adhered to the substrate. The substrate is removed from the lysis solution upon completion of this agitation. The resulting lysis solution is incubated and then genomic DNA from the lysis solution is isolated to form a gDNA solution. From this, identity of at least two nucleotides present in the human TGFβI gene is determined using at least two oligonucleotide primer pairs and the gDNA solution. These at least two nucleotides are located at respective independent positions of the TGFβI gene corresponding to respective independent single nucleotide polymorphisms (SNPs) associated with corneal dystrophy.

1 .- 19 . (canceled) 
     
     
         20 . A reaction mixture for detecting corneal dystrophy in a human subject, the reaction mixture comprising:
 (A) at least a first amplification primer pair for amplifying and determining a first TGFβI gene sequence from a biological sample from the subject; and   (B) a first set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:50.   
     
     
         21 . The reaction mixture according to  claim 20 , further comprising a second set of detection probes including a fourth detection probe comprising the sequence of SEQ ID NO:25 and one or two detection probes selected from a group consisting of a fifth detection probe comprising the sequence of SEQ ID NO:26, a sixth detection probe comprising the sequence of SEQ ID NO:48 and a seventh detection probe comprising the sequence of SEQ ID NO:49, and at least a second amplification primer pair for amplifying and determining a second TGFβI gene sequence from the biological sample from the subject. 
     
     
         22 . The reaction mixture according to  claim 20 , wherein the first amplification primer pair comprises a first amplification primer and a second amplification primer, the first amplification primer comprises nucleotide sequence SEQ ID NO:43, and the second amplification primer comprises nucleotide sequence SEQ ID NO:44. 
     
     
         23 . The reaction mixture according to  claim 20 , wherein the first TGFβI gene sequence comprises nucleotides encoding amino acid residue 555 from the biological sample from the subject. 
     
     
         24 . The reaction mixture according to  claim 21 , wherein the first TGFβI gene sequence comprises nucleotides encoding amino acid residue 555 from the biological sample from the subject and the second TGFβI gene sequence comprises nucleotides encoding amino acid residue 124 from the biological sample from the subject. 
     
     
         25 . The reaction mixture according to  claim 24 , wherein the first amplification primer pair comprises a first amplification primer and a second amplification primer, the first amplification primer comprises nucleotide sequence SEQ ID NO:43, and the second amplification primer comprises nucleotide sequence SEQ ID NO:44, and the second amplification primer pair comprises a third amplification primer and a fourth amplification primer, the third amplification primer comprises a nucleotide sequence SEQ ID NO:1, and the fourth amplification primer comprises a nucleotide sequence SEQ ID NO:2. 
     
     
         26 . A method for detecting whether or not a human subject carries a mutation that causes corneal dystrophy comprising:
 (A) amplifying a first TGFβI gene sequence from a biological sample from the subject using a reaction mixture comprising at least a first amplification primer pair for amplifying a TGFβI gene sequence and a set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:50;   (B) hybridizing one detection probe of the set of at least three detection probes to the first TGFβI gene sequence; and   (C) detecting whether or not a mutation in the first TGFβI gene sequence based on (i) the hybridization of the one detection probe of the set of at least three detection probes that has hybridized in step (B) to the first TGFβI gene sequence and (ii) the failure of the other two detection probes of the set of at least three detection probes to hybridize to the first TGFβI gene sequence.   
     
     
         27 . The method according to  claim 26 , wherein the reaction mixture comprises:
 (A) at least a first amplification primer pair for amplifying and determining a first TGFβI gene sequence from a biological sample from the subject; and   (B) a first set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:5   
     
     
         28 . A method for detecting whether or not a human subject carries a genomic mutation associated with corneal dystrophy in a sample from the subject, the method comprising:
 (A) using epithelial cells of the subject adhered to a tip of a substrate;   (B) agitating the tip of the substrate in a lysis solution that lyses cells adhered to the substrate;   (C) removing the substrate from the lysis solution upon completion of the agitating (B);   (D) incubating the lysis solution after the removing (C);   (E) isolating genomic DNA from the lysis solution to form a gDNA solution; and   (F) determining an identity of at least a nucleotide present in the TGFβI gene using at least a first primer pair for amplifying a TGFβI gene sequence, a first set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:50, and the gDNA solution by concurrently exposing the gDNA solution to the first set of at least three detection probes, wherein:
 the at least a nucleotide is located at a particular position of the TGFβI gene corresponding to a single nucleotide polymorphism (SNP) associated with corneal dystrophy. 
   
     
     
         29 . The method according to  claim 28 , further comprising:
 (G) determining an identity of at least a second nucleotide present in the TGFβI gene using at least a second primer pair, a second set of detection probes including a fourth detection probe comprising the sequence of SEQ ID NO:25 and one or two detection probes selected from a group consisting of a fifth detection probe comprising the sequence of SEQ ID NO:26, a sixth detection probe comprising the sequence of SEQ ID NO:48 and a seventh detection probe consisting of the sequence of SEQ ID NO:49, and the gDNA solution by concurrently exposing the gDNA solution to the second set of detection probes, wherein:
 the at least second nucleotide is located at a second particular position, independent of the position of the at least a nucleotide, of the TGFβI gene corresponding to a second single nucleotide polymorphism (SNP) associated with corneal dystrophy.