ISOTHERMAL REAL-TIME PCR METHOD FOR DETERMINING PRESENCE OF A PRE-DETERMINED NUCLEIC ACID SEQUENCE IN HUMAN SAMPLES

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 an elongated DNA sequence is present in the sample, wherein presence of the elongated DNA sequence in the sample is indicative of the presence of the pre-determined nucleic acid sequence in the sample, wherein the sample is obtained from a human subject 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 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; (c) incubating the sample resulting from steps (a) and (b) at a fixed temperature; (d) determining whether an elongated DNA sequence is present in the sample, wherein presence of the elongated DNA sequence in the sample is indicative of the presence of the pre-determined nucleic acid sequence in the sample wherein the sample is obtained from a human subject and wherein no F3 primer is used.

2. The method of claim 1, wherein four of the at least five primers are 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 fifth primer is 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 4, wherein the pre-determined RNA or DNA sequence is comprised in a pathogen.

6. The method of claim 5, wherein the pathogen is a virus, a bacterium, a fungus or a parasite.

7. The method of claim 6, wherein the pathogen is a Human herpesvirus or a bacterium of the genus Mycoplasma.

8. The method of any one of claims 1 to 7, wherein the fixed temperature is between 50 and 75° C.

9. The method of any one of claims 1 to 8, wherein the sample in step (c) is incubated for 1 to 120 minutes.

10. The method of any one of claims 1 to 9, 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.

11. An anti-infective composition for use in the treatment of an infection of a pathogen, wherein the subject has previously been determined to be infected by the pathogen using the method of any one of claims 1 to 10.

12. The anti-infective composition for use of claim 11, wherein the pathogen is a virus, a bacterium, a fungus or a parasite.

13. The anti-infective composition for use of claim 11 or 12, wherein the anti-infective composition comprises an antiviral, antibiotic, antifungal or antiparasitic drug, respectively.

14. The anti-infective composition for use of any one of claims 12 to 13, wherein the pathogen is a Human herpesvirus or a bacterium of the genus Mycoplasma.

Description

[0114] While aspects of the invention are illustrated and described in detail in the figures and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope and spirit of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below.

[0115] FIG. 1 shows a comparison of the five and six primer system for detecting a pre-determined 16S rRNA sequence in a bacterium of the Mollicutes class using 5 or 6 primers and the methods provided herein.

[0116] FIG. 2 shows a comparison of the five and six primer system for detecting a pre-determined DNA sequence in a Human herpesvirus 1 using 5 or 6 primers and the methods provided herein.

[0117] Furthermore, in the claims the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single unit may fulfill the functions of several features recited in the claims. The terms “essentially”, “about”, “approximately” and the like in connection with an attribute or a value particularly also define exactly the attribute or exactly the value, respectively. Any reference signs in the claims should not be construed as limiting the scope.

EXAMPLES

[0118] 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.

[0119] The Novel 5 Primer System without F3 Amplifies Mollicutes as Efficient as 6 Primer System with F3

TABLE-US-00003 TABLE 1 Primers FIP TGC GGG TCC LPF GTT TGA GTT  CCG TCA ATT TCA TTC TTG  GCC TGG GTA (SEQ ID NO: 3)  GTA CAT TCG (SEQ ID NO: 1) BIP CAA GTG GTG LPB CTT AAT TCG  GAG CAT GTT ACG GTA CAC  TGT CAA GTC (SEQ ID NO: 4)  TAG GTA AGG (SEQ ID NO: 2) B3 TGT TTC CAT F3 GTT AAC ACA  AAC TTT GCC TTA AGT ATC  (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 Add 17.0 μl PEM per reaction

[0120] Template Addition

[0121] Add 8.0 μl extracted RNA

[0122] 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 Cycles Temperature Acquisition Time Ramp rate Amplification 25 65° C. None 27 s 4.4° C. Single 30 s 4.4° C. Quant Melt Integration Channel Dye Factor Factor Time Dye #1, SYBR 20.00 1.2 Dynamic acquisition 470/514 Green I

[0123] The Novel 5 Primer System without F3 Amplifies Human Herpesvirus 1 as Efficient as 6 Primer System with F3

TABLE-US-00008 TABLE 6 Primers FIP GTT GGG TGG LPF TTG GTG GGA TGG AGG AGA ACC CCC GAT CGT CCT TTT AC (SEQ ID NO 9) GGT TCT TGT CGG T (SEQ ID NO: 7) BIP GGT CGT CCC LPB AAC ATG ACC TCG CAT GAA CAG ACC GCG GCG TGG GGC AC TAA GGC TGA TG (SEQ ID NO: 10) (SEQ ID NO: 8) B3 TAC TTG GCA F3 GCC GTT GTT TGG GGG GTG CCC ATT ATC (SEQ ID NO: 11) CC (SEQ ID NO: 12)

TABLE-US-00009 TABLE 7 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-00010 TABLE 8 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-00011 TABLE 9 Primer/Enzyme mix (PEM) Vol/rx Isothermal master mix 15.0 μl Primer mix  2.0 μl 17.0 μl Add 17.0 μl PEM per reaction

[0124] Template Addition

[0125] Add 8.0 μl extracted DNA

[0126] Add 8.0 μl RNase-free H2O as negative assay control

TABLE-US-00012 TABLE 10 Settings for isothermal amplification and dye acquisition Cycles Temperature Acquisition Time Ramp rate Amplification 25 65° C. None 27 s 4.4° C. Single 30 s 4.4° C. Quant Melt Integration Channel Dye Factor Factor Time Dye #1, SYBR 20.00 1.2 Dynamic acquisition 470/514 Green I