MULTI-PRIMER ASSAY FOR MYCOPLASMA DETECTION
Disclosed is a multi-primer amplification assay, method and kits for detecting Mycoplasma species and closely related species utilizing a plurality of oligonucleotide primers in contact with a sample in a single vessel and detecting the amplification product, wherein the presence of an amplification product indicates Mycoplasma in the sample.
1 . A multi-primer assay for detecting Mycoplasma comprising:
a.) contacting a sample with the plurality of oligonucleotide primers selected from the group consisting of SEQ ID NOs:1-144 in a single vessel;
b.) performing a multi-primer amplification reaction in the vessel, wherein each of the plurality of oligonucleotide primers is present for participation in amplifying a target nucleic acid in the sample to produce an amplification product; and
c.) detecting the amplification product, wherein the presence of an amplification product indicates Mycoplasma in the sample.
2 . The assay according to claim 1 , wherein at least one of the oligonucleotide primers comprises a modified nucleobase at the third nucleobase from the 3′ end of the primer.
3 . The assay according to claim 1 , wherein at least one of the oligonucleotide primers comprises a modified nucleobase at the second nucleobase from the 3′ end of the primer.
4 . The assay of claim 1 wherein the plurality of primers comprises at least 15 forward primers and at least 16 reverse primers.
5 . The assay according to claim 1 , wherein at least twenty oligonucleotide primers are present in the multi-primer amplification reaction.
6 - 8 . (canceled)
9 . The assay according to claim 1 , wherein the sample is selected from a cell culture, a tissue culture, a food sample, an environmental sample, an agricultural sample, a biopharmaceutical sample, a pharmaceutical sample, or water.
10 - 11 . (canceled)
12 . The assay according to claim 2 , wherein the modification averts primer-dimer formation in the amplification reaction.
13 . The assay according to claim 3 , wherein the modification averts primer-dimer formation in the amplification reaction.
14 . The assay according to claim 1 , wherein the contacting comprises hybridizing one or more of the oligonucleotide primers to a target nucleic acid in the sample.
15 . The assay according to claim 14 , wherein the target nucleic acid is extracted from the sample prior to hybridizing with the one or more oligonucleotide primers.
16 . The assay according to claim 1 , wherein the detecting comprises a detector selected from the group consisting of a nucleic acid dye, a reporter probe, or a reporter probe and a nucleic acid dye.
17 . The assay according to claim 1 , wherein the assay comprises a discriminatory positive control (DPC) nucleic acid.
18 . The assay according to claim 17 , wherein the DPC nucleic acid is added to the sample prior to extraction of the target nucleic acid of the sample.
19 . The assay according to claim 18 , wherein the DPC nucleic acid is extracted simultaneously with the target nucleic acid of the sample.
20 . The assay according to claim 19 , wherein the DPC nucleic acid is amplified in the multi-primer amplification reaction.
21 . The assay according to claim 20 , wherein the DPC amplification product is detected.
22 . The assay according to claim 21 , wherein detection of the DPC amplification product and no detection of the Mycoplasma amplification product is a confirmed negative assay for Mycoplasma.
23 . The assay according to claim 21 , wherein detection of the DPC amplification product and detection of the Mycoplasma amplification product is a confirmed positive assay for Mycoplasma.
24 - 25 . (canceled)
26 . The assay according to claim 21 , wherein the detecting comprises a melting temperature for the discriminatory positive control distinguishable from a melting temperature for the Mycoplasma.
27 . The assay according to claim 1 , wherein the assay detects more than 90 Mycoplasma species as well as related microorganisms Acholeplama laidlwaii and Spiroplasma citri.
28 . A method for detecting Mycoplasma comprising:
a.) extracting nucleic acid from a sample to be tested for Mycoplasma;
b.) combining in a single vessel a plurality of oligonucleotide primers and the nucleic acid from the sample;
c.) contacting the nucleic acid from the sample with the plurality of oligonucleotide primers in the vessel;
d.) performing a multi-primer amplification reaction in the vessel, wherein each of the plurality of oligonucleotide primers is present for participation in amplifying the sample to produce an amplification product; and
e.) detecting the amplification product, wherein the presence of the amplification product indicates Mycoplasma in the sample.
29 . The method according to claim 28 , wherein the plurality of oligonucleotide primers is selected from the group consisting of SEQ ID NOS:1-144.
30 - 33 . (canceled)
34 . A kit for detecting Mycoplasma comprising a container having disposed therein a plurality of oligonucleotide primers selected from the group consisting of SEQ ID NOs: 1-144.
35 . The kit of claim 34 , further comprising one or more of a polymerase, a detectable reporter, a protocol and a discriminating positive control nucleic acid.
36 . The kit of claim 35 , wherein the detectable reporter is selected from the group consisting of a nucleic acid dye, a reporter probe or a reporter probe and a nucleic acid dye.