Multi-primer assay for
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 in a sample, comprising:
a.) contacting the 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 in the presence of a nucleic acid dye or a dye probe, 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 comprising the nucleic acid dye or the dye probe; and
c.) detecting a signal from the nucleic acid dye or the dye probe of the amplification product, wherein the presence of an amplification product indicates Mycoplasma in the sample;
wherein the assay is capable of detecting more than 90 Mycoplasma species or related microorganisms Acholeplama laidlwaii and Spiroplasma citri.
2. The assay according to claim 1 , wherein at least one of the plurality of oligonucleotide primers comprises a modified nucleobase at the third or fourth nucleobase from the 3′ end of the primer.
3. The assay of claim 1 wherein the plurality of primers comprises at least 15 forward primers and at least 16 reverse primers.
4. The assay according to claim 1 , wherein the plurality of oligonucleotide primers comprises at least twenty oligonucleotide primers.
5. The assay according to claim 1 , wherein the sample comprises either a cell culture, a tissue culture, a food sample, an environmental sample, an agricultural sample, a biopharmaceutical sample, a pharmaceutical sample, or water.
6. 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.
7. The assay according to claim 6 , wherein the target nucleic acid is extracted from the sample prior to hybridizing with the one or more oligonucleotide primers.
8. 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.
9. The assay according to claim 1 , wherein the assay comprises a discriminatory positive control (DPC) nucleic acid.
10. The assay according to claim 9 , wherein the DPC nucleic acid is added to the sample prior to extraction of the target nucleic acid of the sample.
11. The assay according to claim 10 , wherein the DPC nucleic acid is extracted simultaneously with the target nucleic acid of the sample.
12. The assay according to claim 11 , wherein the DPC nucleic acid is amplified in the multi-primer amplification reaction and the DPC amplification product is detected.
13. The assay according to claim 12 , wherein the detecting comprises a melting temperature for the discriminatory positive control distinguishable from a melting temperature for the Mycoplasma.
14. The assay according to claim 13 , wherein detection of the DPC amplification product and no detection of the Mycoplasma amplification product is a confirmed negative assay for Mycoplasma ; wherein detection of the DPC amplification product and detection of the Mycoplasma amplification product is a confirmed positive assay for Mycoplasma.
15. 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, said oligonucleotide primers selected from the group consisting of SEQ ID NOS:1-144;
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;
wherein the assay can detect more than 90 Mycoplasma species or related microorganisms Acholeplama laidlwaii and Spiroplasma citri.
16. The method for detecting Mycoplasma of claim 15 comprising:
a.) optionally, combining a discriminating positive control (DPC) nucleic acid with the sample to be tested for Mycoplasma ;
b.) extracting nucleic acid from the combined sample and DPC.
17. The method for detecting Mycoplasma of claim 15 , wherein a DPC nucleic acid is added to the vessel.
18. The method for detecting Mycoplasma of claim 17 , wherein the DPC nucleic acid is amplified in the multi-primer amplification reaction and the DPC amplification product is detected.
19. The method for detecting Mycoplasma of claim 18 , wherein the detecting comprises a melting temperature for the discriminatory positive control distinguishable from a melting temperature for the Mycoplasma.