POLYNUCLEOTIDES FOR THE AMPLIFICATION AND DETECTION OF NEISSERIA GONORRHOEAE
Disclosed herein are primers and probes related to the detection of Neisseria gonorrhoeae via nucleic acid amplification testing (NAAT), for example to amplify and determine the presence of N. gonorrhoeae nucleic acids present in test samples. Specifically the present disclosure describes primers and probes that bind to the small subunit rRNA (cytosine (967)-C(5))-methyltransferase or rsmB gene of N. gonorrhoeae for detection via loop mediated isothermal amplification (LAMP) and molecular beacon hybridization.
1 . A composition comprising a set of polynucleotides selected from the group consisting of Set-1 through Set-55.
2 . The composition of claim 1 , further comprising a probe.
3 .- 13 . (canceled)
14 . A molecular beacon comprising a fluorophore, a quencher and a polynucleotide, wherein the polynucleotide is selected from the group consisting of: SEQ ID NO: 35, SEQ ID NO: 36. SEQ ID NO: 69 through SEQ ID NO: 72, and SEQ ID NO:
92 through SEQ ID NO: 101.
15 .- 16 . (canceled)
17 . A method of detecting Neisseria gonorrhoeae in a test sample, the method comprising:
a. extracting nucleic acid from the test sample;
b. amplifying a target sequence by reacting the nucleic acid extracted in step (a) with a reaction mixture comprising a strand displacement DNA polymerase and a sequence-specific primer set, wherein said sequence-specific primer set is selected from the group consisting of Set-1 through Set-55; and
c. detecting the presence or absence of an amplified product of step (b); wherein the presence of said amplification product is indicative of the presence of Neisseria gonorrhoeae in the test sample.
18 . The method of claim 17 , wherein the amplification in step (b) of the target sequence is performed at between about 60° C. and 67° C. for less than 30 minutes.
19 . The method of claim 17 , wherein the amplification step is performed for less than 15 minutes.
20 . The method of claim 19 , wherein the amplification step is performed for less than nine minutes.
21 . The method of claim 17 , wherein detecting the presence or absence of the amplification product comprises hybridizing the amplified product with a probe comprising a polynucleotide attached to a label.
22 . The method of claim 21 , wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 72 (MB6), SEQ ID NO: 94 (MB9), and SEQ ID NO: 101 (MB16), and the sequence-specific primer set is selected from the group consisting of Set-8, Set-28, and Set-45.
23 . The method of claim 21 , wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 69 (MB3), SEQ ID NO: 70 (MB4), SEQ ID NO: 71 (MB5), SEQ ID NO: 95 (MB10), SEQ ID NO: 96 (MB11), SEQ ID NO: 97 (MB12), and SEQ ID NO: 98 (MB13), and the sequence-specific primer set is selected from the group consisting of Set-9, Set-10, Set-11, Set-12, Set-29, Set-30, Set-31, Set-32, Set-46, Set-47, Set-48 and Set-49.
24 . The method of claim 21 , wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 99 (MB14) and SEQ ID NO: 100 (MB15), and the sequence-specific primer set is selected from the group consisting of Set-57, Set-58, Set-59, Set-60, Set-61, Set-62, Set-64, Set-65, Set-66, Set-67, set-68, Set-69, Set-71, Set-72, Set-73, Set-74, Set-75, and Set-76.
25 . The method of claim 21 , wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 35 (MB1), SEQ ID NO:36 (MB2), SEQ ID NO: 92 (MB7), and SEQ ID NO: 93 (MB8), and the sequence-specific primer set is selected from the group consisting of Set-1, Set-2, Set-3, Set-4, Set-5, Set-6, Set-19, Set-20, Set-21, Set-22, Set-23, Set-24, Set-25, Set-26, Set-39, Set-40, Set-41, Set-42, Set-43, Set-56, Set-63, and Set-70.
26 . The method of claim 21 , wherein the probe is a molecular beacon.
27 . The method of claim 17 , wherein the reaction mixture further comprises a reverse transcriptase.
28 . The method of claim 17 , wherein Neisseria gonorrhoeae is present in the test sample at a concentration of ≤100 CFU/mL.
29 . The method of claim 28 , wherein Neisseria gonorrhoeae is present in the test sample at a concentration of ≤10 CFU/mL.
30 . A kit comprising the composition of claim 1 and amplification reagents.
31 . The kit of claim 30 , wherein the amplification reagents comprise a strand displacement polymerase.
32 . The kit of claim 30 , further comprising a probe.
33 . A method of detecting Neisseria gonorrhoeae in a test sample, the method comprising:
a. extracting nucleic acid from the test sample;
b. amplifying a target sequence by reacting the nucleic acid extracted in step (a) for less than twenty minutes with a reaction mixture comprising a strand displacement DNA polymerase and a sequence-specific LAMP primer set; and
c. detecting the presence or absence of an amplified product of step (b); wherein the presence of said amplification product is indicative of the presence of Neisseria gonorrhoeae in the test sample.
34 .- 41 . (canceled)