ISOTHERMAL REAL-TIME PCR METHOD FOR DETERMINING PRESENCE OF A PRE-DETERMINED NUCLEIC ACID SEQUENCE IN ANIMAL 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 an animal 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 an animal 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 claims 1 and 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 embodiment 6, wherein the pathogen is Streptococcus equi equi, Equine herpes virus, in particular Equine herpesvirus 1 or 4, or Equine influenza virus

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. A method of treating an animal infected by a pathogen, the method comprising administering to the animal an efficient amount of a therapeutic drug, wherein the animal has previously been determined to be infected by the pathogen using the method of any one of claims 1 to 10.

12. The method of claim 11 wherein the animal is a horse.

13. The method of claim 11 or 12, wherein the pathogen is a virus, a bacterium, a fungus or a parasite.

14. The method of any one of claim 11 or 13, wherein the therapeutic drug is an anti-viral, antibiotic, antifungal or antiparasitic drug, respectively.

15. The method of any one of claims 11 to 14, wherein the pathogen is Streptococcus equi equi, Equine herpes virus, in particular Equine herpesvirus 1 or 4, or Equine influenza virus.

Description

EXAMPLES

[0113] 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.

The Novel 5 Primer System without F3 Amplifies Equine influenza Virus RNA as Efficient as the 6 Primer System with F3

[0114]

TABLE-US-00005 TABLE 1 Primers FIP CAA GTC TCT GCG CGA TCT AGG LPF TTT GAG GGG GCC TGA TGG TCG AAA CGT ACG TTC (SEQ ID (SEQ ID NO: 3) NO: 1) BIP GAA GAT GTC TTT GCA GGG TTG LPB ACC GAT CTT GAG GCA CTC GTC TTG TCT TTA GCC (SEQ ID (SEQ ID NO: 4) NO: 2) B3 AAT CCC TTT AGT YAG AG (SEQ F3 AGT CTT CTG ACC GAG GTC ID NO: 5) (SEQ ID NO: 6)

TABLE-US-00006 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-00007 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-00008 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

Template Addition

[0115] Add 8.0 μl of template RNA

[0116] Add 8.0 μl RNase-free H2O as negative assay control

TABLE-US-00009 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 acquisition #1,470/514 SYBR 20.00 1.2 Dynamic Green I

The Novel 5 Primer System without F3 Amplifies Equine herpes Virus Type 1 DNA as Efficient as 6 Primer System with F3

[0117]

TABLE-US-00010 TABLE 6 Primers FIP GTC GTA RAA CCT GAG AGC LPF GAG CTG GTT GCG GCG CTG GGC CTG CTA GAC TAC AGC (SEQ ID NO: 9) (SEQ ID NO: 7) BIP CGA CAG CGT GGT CAA CGT LPB ATA CCG CAG TGA TTA TGC TGA AAA AGC TGG CGA TCC (SEQ ID NO: 10) (SEQ ID NO: 8) B3 TCC CCC ACT TTA CCC AG F3 CTG AGC TGG AGG ACA C (SEQ ID NO: 11) (SEQ ID NO: 12)

TABLE-US-00011 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-00012 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-00013 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

Template Addition

[0118] Add 8.0 μl template DNA

[0119] Add 8.0 μl RNase-free H2O as negative assay control

TABLE-US-00014 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 acquisition #1,470/514 SYBR 20.00 1.2 Dynamic Green I