Amplification of target nucleic acids using gap filling ligase chain reaction

A method for detecting the presence of a target nucleic acid in a sample, said method employing a ligase chain reaction to create geometrically increasing numbers of reorganized probe molecules in the presence of said target nucleic acid, said method comprising:

• a) providing a sample suspected to contain nucleic acid, the nucleic acid having a target sequence of the formula:
• 5'-(N).sub.h LE.sub.p E'.sub.q L(N).sub.k -3' or its complement 3'-(N').sub.h L'E'.sub.p E.sub.q J'(N').sub.k -5',
• wherein E represents any base, E' is the complement of E, p and q are independently integers from 1 to about 10, L represents any base except E, L' is the complement of L, J represents any base except E', J' is the complement of J, N represents any base, and h and k are independently integers from 5 to 100, provided (p+h) and (q+k) each are greater than 10, wherein (N).sub.h and (N).sub.k represent sequences that are characteristic for the target sought to be detected;
• b) providing a plurality of each of four probes having the formulas:
• 5'-(N).sub.h L E.sub.p -3' A
• 3'-(N').sub.h L'-5' A'
• 5'-J(N).sub.k -3' B
• 3'-E'.sub.q J'(N').sub.k -5' B'
• wherein N, N', L, L', E, E', J, J'h, p, k and q are defined above, except that A and A' or B and B' need not be identical in length, and wherein at least one of the probes is labeled with a moiety capable of detection;
• and also providing deoxyribonucleotide triphosphates of E', a polymerase reagent and a ligase reagent;
• c) performing the following cycle at least once:
• i) mixing said probes with said sample under hybridizing conditions to allow probes to hybridize to the target sequence and its complement if present;
• ii) using target sequence or reorganized probes created therefrom as template, extending probes A and B' with said polymerase reagent by adding E' deoxyribonucleotide triphosphates to their 3' ends
• iii) ligating extended probe A to probe B, and extended probe B' to probe A', using said ligase reagent to form reorganized probe molecules;
• and
• iv) providing denaturing conditions to separate said reorganized probe molecules from said template;
• d) separating reorganized probe molecules from unreorganized labeled probes;
• and
• e) detecting the presence of said labeled probes in the reorganized fraction as a measure of the presence of the target sequence.

A method for determining the presence of HIV 1 in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 1667, SEQ ID Nos 1 ,2 ,3 and 4;
• at map position 912, SEQ ID Nos. 5 ,6 ,7 and 8;
• at map position 2086, SEQ ID Nos. 9, 10, 11 and 12;
• at map position 789, SEQ ID Nos. 13, 14, 15 and 16;
• at map position 508, SEQ ID Nos. 17, 18, 19 and 20;
• at map position 5569, SEQ ID Nos. 21, 22, 23 and 24;
• at map position 1450, SEQ ID Nos. 25, 26, 27 and 28;
• at map position 1573, SEQ ID Nos. 29, 30, 31 and 32;
• at map position 4060, SEQ ID Nos. 33, 34, 35 and 36;
• at map position 2683, SEQ ID Nos. 37, 38, 39 and 40;
• at map position 1753, SEQ ID Nos. 41, 42, 43 and 44;
• and
• at map position 3697, SEQ ID Nos. 45, 46, 47 and 48.

A method for determining the presence of HIV 2 in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 2060, SEQ ID Nos 49, 50, 51 and 52;
• at map position 4661, SEQ ID Nos. 53, 54, 55 and 56;
• and
• at map position 1094, SEQ ID Nos. 57, 58, 59 and 60.

A method for determining the presence of HPV in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 631, SEQ ID Nos 61, 62, 63 and 64;
• at map position 480, SEQ ID Nos 65, 66, 67 and 68;
• at map position 488, SEQ ID Nos. 69, 70, 71 and 72;
• and
• at map position 6604, SEQ ID Nos. 73, 74, 75 and 76.

A method for determining the presence of Chlamydia trachomatis in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 36, SEQ ID Nos 85, 86, 87 and 88;
• at map position 552, SEQ ID Nos. 89, 90, 91 and 92;
• and
• at map position 6693, SEQ ID Nos. 93, 94, 95 and 96.

A method for determining the presence of Neisseria gonorrhoeae, in a sample, said method comprising amplifying a target region using at least two probes specific for said region and detecting amplified product, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 27, SEQ ID Nos 97, 98, 99 and 100;
• at map position 66, SEQ ID Nos 101, 102, 103 and 104;
• at map position 114, SEQ ID Nos 105, 106, 107 and 108;
• at map position 822, SEQ ID Nos. 109, 110, 111 and 112;
• and
• at map position 933, SEQ ID Nos. 113, 114, 115 and 116.

A composition of matter useful for detecting HIV 1, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 1667, SEQ ID Nos 1, 2, 3 and 4;
• at map position 912, SEQ ID Nos. 5, 6, 7 and 8;
• at map position 2086, SEQ ID Nos. 9, 10, 11 and 12;
• at map position 789, SEQ ID Nos. 13, 14, 15 and 16;
• at map position 508, SEQ ID Nos. 17, 18, 19 and 20;
• at map position 5569, SEQ ID Nos. 21, 22, 23 and 24;
• at map position 1450, SEQ ID Nos. 25, 26, 27 and 28;
• at map position 1573, SEQ ID Nos. 29, 30, 31 and 32;
• at map position 4060, SEQ ID Nos. 33, 34, 35 and 36;
• at map position 2683, SEQ ID Nos. 37, 38, 39 and 40;
• at map position 1753, SEQ ID Nos. 41, 42, 43 and 44;
• and
• at map position 3697, SEQ ID Nos. 45, 46, 47 and 48.

A composition of matter useful for detecting HIV 2, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe set consisting of:

• at map position 2060, SEQ ID Nos 49, 50, 51 and 52;
• at map position 4661, SEQ ID Nos. 53, 54, 55 and 56;
• and
• at map position 1094, SEQ ID Nos. 57, 58, 59 and 60.

A composition of matter useful for detecting HPV, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe set consisting of:

• at map position 631, SEQ ID Nos 61, 62, 63 and 64;
• at map position 480, SEQ ID Nos 65, 66, 67 and 68;
• at map position 488, SEQ ID Nos. 69, 70, 71 and 72;
• and
• at map position 6604, SEQ ID Nos. 73, 74, 75 and 76.

A composition of matter useful for detecting Chlamydia trachomatis, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 36, SEQ ID Nos 85, 86, 87 and 88;
• at map position 552, SEQ ID Nos. 89, 90, 91 and 92;
• and
• at map position 6693, SEQ ID Nos. 93, 94, 95 and 96.

A composition of matter useful for detecting Neisseria gonorrhoeae, said composition comprising a mixture of at least two probes, wherein said at least two probes are each selected from a single probe set, said probe set being selected from the group of probe sets consisting of:

• at map position 27, SEQ ID Nos 97, 98, 99 and 100;
• at map position 66, SEQ ID Nos 101, 102, 103 and 104;
• at map position 114, SEQ ID Nos 105, 106, 107 and 108;
• at map position 822, SEQ ID Nos. 109, 110, 111 and 112;
• and
• at map position 933, SEQ ID Nos. 113, 114, 115 and 116.