ISOTHERMAL REAL-TIME PCR METHOD FOR DETERMINING PRESENCE OF A PRE-DETERMINED NUCLEIC ACID SEQUENCE OF A BACTERIUM OF THE MOLLICUTES CLASS IN A SAMPLE

Abstract

The present invention relates to a method for determining presence of a pre-determined nucleic acid sequence in a sample, the method comprising the steps of adding one or more enzyme(s) providing activities of RNA- and/or DNA-dependent DNA polymerase activity and strand-displacement activity to the sample to be analysed for the presence of the pre-determined nucleic acid sequence; adding at least five DNA primers to the sample to be analysed for the presence of the pre-determined nucleic acid sequence, wherein at least one DNA primer comprises a sequence hybridisable to the nucleic acid sequence and at least one DNA primer comprises a sequence hybridisable to the DNA sequence reverse-complementary to the nucleic acid sequence; incubating the sample resulting at a fixed temperature; determining whether a double-stranded elongated DNA sequence is present in the sample, wherein presence of the double-stranded elongated DNA sequence in the sample is indicative of the presence of the pre-determined nucleic acid sequence in the sample, wherein the pre-determined nucleic acid sequence is of a bacterium of the Mollicutes class and wherein no F3 primer is used.

Claims

1. A method for determining presence of a pre-determined nucleic acid sequence in a sample, the method comprising the steps of: (a) adding one or more enzyme(s) providing activities of RNA- and/or DNA-dependent DNA polymerase activity and strand-displacement activity to the sample to be analysed for the presence of the pre-determined nucleic acid sequence; (b) adding at least five DNA primers to the sample to be analysed for the presence of the pre-determined nucleic acid sequence, wherein at least one DNA primers comprises a sequence hybridisable to the nucleic acid sequence and at least one DNA primers comprise a sequence hybridisable to the DNA sequence reverse-complementary to the nucleic acid sequence; (c) incubating the sample resulting from steps (a) and (b) at a fixed temperature; (d) determining whether a double-stranded elongated DNA sequence is present in the sample, wherein presence of the double-stranded elongated DNA sequence in the sample is indicative of the presence of the pre-determined nucleic acid sequence in the sample wherein the pre-determined nucleic acid sequence is of a bacterium of the Mollicutes class and wherein no F3 primer is used.

2. The method of claim 1, wherein the at least five primers comprise a forward inner primer (FIP), backward inner primer (BIP), loop primer forward (LPF) and loop primer backwards (LPB), respectively.

3. The method of claim 1 or 2, wherein the at least five primers further comprise a B3 primer.

4. The method of any one of claims 1 to 3, wherein the pre-determined nucleic acid sequence is an RNA or DNA sequence.

5. The method of any one of claims 1 to 5, wherein the bacterium of the Mollicutes class is of the genus Mycoplasma, Spiroplasma, Acholeplasma, or Ureaplasma.

6. The method of claim 6, wherein the bacterium is M. orale, M. arginini, M. fermentans, M. hyorhinis, A. laidlawii, M. hominis, M. synoviae, S. citri, M. pneumoniae, M. bovis, M. sahvarium or M. gallisepticum.

7. The method of any one of claims 4 to 6, wherein the RNA is comprised in 16S rRNA or 23S rRNA.

8. The method of any one of claims 4 to 6, wherein the DNA is the gene coding for 16S rRNA or the gene coding for 23 S rRNA.

9. The method of any one of claims 1 to 8, wherein the sample is obtained from primary- or modified cells and/or tissues, cell cultures, culture medium and/or additives, cell derived products, laboratory equipment or biopharmaceutical products such as ATMPs.

10. The method of any one of claims 1 to 9, wherein the fixed temperature is between 50 and 75° C.

11. The method of any one of claims 1 to 10, wherein the sample in step (c) is incubated for 1 to 120 minutes.

12. The method of any one of claims 1 to 11, wherein presence of the double-stranded elongated DNA sequence in the sample is determined by using a nucleic acid molecule hybridisable to the double-stranded elongated DNA sequence, in particular wherein the nucleic acid molecule is labelled, using a molecule that intercalates in the double-stranded elongated DNA sequence or using turbidity measurement.

13. A method of decontaminating a cell and/or tissue culture infected by a bacterium of the Mollicutes class, the method comprising administering to the culture an efficient amount of an antibiotic, wherein the culture has previously been determined to be infected by a bacterium of the Mollicutes class using the method of any one of claims 1 to 12.

14. The method of claim 13, wherein the antibiotic drug is BM Cyclin.

Description

EXAMPLES

[0105] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0106] The Novel 5 Primer System without F3 Amplifies Mollicutes as Efficient as 6 Primer System with F3

TABLE-US-00003 TABLE 1 Primers FIP TCA TCG TTT ACA GCG TGG ACG AAA LPF CTA CCA GGG TAT CTA ATC GCG TGG GGA GCA (SEQ ID NO: 1) (SEQ ID NO: 3) BIP GCA GCT AAC GCA TTA AAT AGT TTC LPB TGA TCC GCC TGA GTA GTA ACT CTT GCG AGC (SEQ ID NO: 2) (SEQ ID NO: 4) B3 CGG GTC CCC GTC AAT TCC F3 CTA TAC TGA CGC TGA GGG (SEQ ID NO: 5) (SEQ ID NO: 6)

TABLE-US-00004 TABLE 2 Primer mix: novel 5 primer system Final concentration. FIP 1.6 μM BIP 1.6 μM LPF 0.8 μM LPB 0.8 μM B3 0.4 μM

TABLE-US-00005 TABLE 3 Primer mix: LAMP 6 primer system Final concentration FIP 1.6 μM BIP 1.6 μM LPF 0.8 μM LPB 0.8 μM B3 0.2 μM F3 0.2 μM

TABLE-US-00006 TABLE 4 Primer/Enzyme mix (PEM) Vol/rx Isothermal master mix 15.0 μl Primer mix  2.0 μl 17.0 μl

[0107] Add 17.0 μl PEM per reaction

[0108] Template Addition

[0109] Add 8.0 μl extracted RNA

[0110] Add 8.0 μl RNase-free H.sub.2O as negative assay control

TABLE-US-00007 TABLE 5 Settings for isothermal amplification and dye acquisition Temper- Acquisi- Ramp Cycles ature tion Time rate Ampli- 25 65° C. None 27 s 4.4° C. fication Single 30 s 4.4° C. Quant Melt Integration Channel Dye Factor Factor Time Dye #1,470/ SYBR 20.00 1.2 Dynamic acquisition 514 Green I