Granted Patent B2

US 12,392,001 · App. 16/991,216 · Granted Aug 19, 2025

Inventors: Lars Peters (Portland, ME); Stephen A. Judice (Portland, ME); Daniel Shaffer (Portland, ME); Breck Parker (Portland, ME)

Assignee: Envirologix Inc.

C12Q1/701C12Q1/6865C12Q2600/158Y02A50/30

Abstract

Claims (153)

1. A method of amplifying a target RNA molecule in a reverse transcriptase and nicking amplification reaction, the method comprising:

(a) contacting a target RNA molecule in a crude sample with a reverse transcriptase primer in the presence of a reverse transcriptase and dNTPs under conditions permissive for cDNA synthesis, thereby generating a cDNA;

(b) contacting the cDNA with

(i) forward and reverse primers, wherein each primer comprises at least one nicking enzyme recognition sequence and a 3′-terminal recognition region which specifically binds the cDNA in the presence of a nicking enzyme, and one or more 2′ modified nucleotides in the recognition region,

(ii) dNTPs, and

(iii) a strand-displacement polymerase under conditions permissive for the isothermal amplification of the cDNA, thereby generating amplicons.

2. The method of claim 1 , wherein the polynucleotide molecule is an Ebola virus (EBOV), human immunodeficiency virus (HIV), dengue virus, influenza B virus, or bovine diarrhea virus polynucleotide.

3. The method of claim 1 , wherein the method comprises a one-step reaction where the reverse transcriptase and strand-displacement polvmerase are included in a single reaction and steps (a) and (b) are carried out at the same time.

4. The method of claim 1 , wherein the reverse transcriptase enzyme and the strand-displacement DNA polymerase are the same or different enzymes.

5. The method of claim 1 , wherein the method is carried out in about 5, 7, 10, 15, 20, 25, or 30 minutes.

6. The method of claim 1 , wherein the primer used in step (a) has the same sequence as a primer used in step (b).

7. The method of claim 1 , wherein the crude sample is a biological sample or an environmental sample.

8. The method of claim 7 , wherein the biological sample is a swab of a mucosal membrane selected from the group consisting of buccal, nasal, eye, rectal, and vaginal or skin.

9. The method of claim 7 , wherein the biological sample is a tissue sample obtained from a subject, necropsy, or culture media.

10. The method of claim 1 , wherein the strand-displacement DNA polymerase is a 5′-exo-derivative selected from the group consisting of Bst DNA polymerase I, Gst DNA polymerase I, Gka DNA polymerase I, Gca DNA polymerase I, Gan DNA polymerase I, Gbo DNA polymerase I, Gsp70 DNA polymerase I, GspT3 DNA polymerase I, Gsp52 DNA polymerase I and/or fragments thereof.

11. The method of claim 1 , wherein the nicking enzyme is one or more of Nt.BstNBI, Nt.BspD6I, Nt.BspQI, Nt.BsmAI, Nt.AlwI, N.Bst9I, or N.BstSEI.

12. The method of claim 1 , wherein the reverse transcriptase is M-MLV RT, AMV RT, or RSV RT.

13. The method of claim 2 , wherein the forward and reverse primers each comprise a nucleotide with a methoxy modification;

wherein the forward and reverse primers for detection of EBOV comprise primers selected from the group consisting of:

Forward primer:

(SEQ ID NO: 1)

GACTCGATATCGAGTCGCTTCCAmCAGTTATCmUmAmCmCmG,

and

Reverse Primer:

(SEQ ID NO: 2)

GACTCGATATCGAGTCGAAATGCmAACGAmCmAmCmCmU;

wherein the forward and reverse primers for detection of HIV comprise primers selected from the group consisting of:

Forward primers:

(SEQ ID NO: 6)

GACTCGATATCGAGTCTGACTAGmCGGAGGmCmTmAmGmAmAmG,

and

(SEQ ID NO: 7)

GACTCGATATCGAGTCTGACTAGmCAGAGGmCmTmAmGmAmAmG,

and

Reverse Primers:

(SEQ ID NO: 8)

GACTCGATATCGAGTCTATTGACmGCTCmTmCmGmCmAmC,

and

(SEQ ID NO: 9)

GACTCGATATCGAGTCTACTGACmGCTCmTmCmGmCmAmC;

wherein the forward and reverse primers for detection of dengue virus comprise primers selected from the group consisting of:

Forward primer:

(SEQ ID NO: 11)

GACTCGATATCGAGTCCAAAAACmAGCATATTmGmAmCmGmC,

and

Reverse Primer:

(SEQ ID NO: 12)

GACTCGATATCGAGTCAGACAGCmAGGATCmTmCmTmGmG,

and

(SEQ ID NO: 13)

GACTCGATATCGAGTCAGACAGCmAGGATCmTmGmTmGmG;

wherein the forward and reverse primers for detection of influenza B virus comprise primers selected from the group consisting of:

Forward primers:

(SEQ ID NO: 15)

GACTCGATATCGAGTCAAATGCAmGATGGTCTCmAmGmCmTmA,

(SEQ ID NO: 16)

GACTCGATATCGAGTCAAATGCAmAATGGTCTCmAmGmCmTmA,

and

(SEQ ID NO: 17)

GACTCGATATCGAGTCAAATGCAmGATGGTTTCmAmGmCmTmA,

and

Reverse Primers:

(SEQ ID NO: 18)

GACTCGATATCGAGTCCTCCTTTmTCCCATTCCATmTmCmAmTmT,

(SEQ ID NO: 19)

GACTCGATATCGAGTCCTCCCTTmTCCCATTCCATmTmCmAmTmT,

and

(SEQ ID NO: 20)

GACTCGATATCGAGTCCTCCTTTmCCCCATTCCATmTmCmAmTmT;

or

wherein the forward and reverse primers for detection of bovine diarrhea virus comprise primers selected from the group consisting of:

Forward primers:

(SEQ ID NO: 22)

GACTCGATATCGAGTCGGCCCACmTGTATTGCTmAmCmTmGmAmAmA,

and

(SEQ ID NO: 23)

GACTCGATATCGAGTCGGCCCACmTGCACTGCTmAmCmTmAmAmAmA,

and

Reverse Primer:

(SEQ ID NO: 24)

GACTCGATATCGAGTCTGTGATCmAACTCCmAmTmGmTmGmCmC,

wherein mN indicates that the base N is a methoxy base.

14. The method of claim 13 , wherein the modified nucleotide is one or more of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-hydroxyl, 2′-alkyl, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-CH 2 —O-2′-bridge, 4′-(CH 2 ) 2-O-2′-bridge, 2′-LNA, and 2′-O—(N-methylcarbamate).

15. A kit for amplifying a polynucleotide molecule in a reverse transcriptase and nicking amplification reaction, the kit comprising:

a) forward and reverse primers each comprising a nucleotide with a methoxy modification;

wherein the forward and reverse primers are for the detection of EBOV and comprise primers selected from the group consisting of:

Forward primer:

(SEQ ID NO: 1)

GACTCGATATCGAGTCGCTTCCAmCAGTTATCmUmAmCmCmG,

and

Reverse Primer:

(SEQ ID NO: 2)

GACTCGATATCGAGTCGAAATGCmAACGAmCmAmCmCmU;

wherein the forward and reverse primers for detection of HIV comprise primers selected from the group consisting of:

Forward primers:

(SEQ ID NO: 6)

GACTCGATATCGAGTCTGACTAGmCGGAGGmCmTmAmGmAmAmG,

and

(SEQ ID NO: 7)

GACTCGATATCGAGTCTGACTAGmCAGAGGmCmTmAmGmAmAmG,

and

Reverse Primers:

(SEQ ID NO: 8)

GACTCGATATCGAGTCTATTGACmGCTCmTmCmGmCmAmC,

and

(SEQ ID NO: 9)

GACTCGATATCGAGTCTACTGACmGCTCmTmCmGmCmAmC;

wherein the forward and reverse primers for detection of dengue virus comprise primers selected from the group consisting of:

Forward primer:

(SEQ ID NO: 11)

GACTCGATATCGAGTCCAAAAACmAGCATATTmGmAmCmGmC,

and

Reverse Primer:

(SEQ ID NO: 12)

GACTCGATATCGAGTCAGACAGCmAGGATCmTmCmTmGmG,

and

(SEQ ID NO: 13)

GACTCGATATCGAGTCAGACAGCmAGGATCmTmGmTmGmG;

wherein the forward and reverse primers for detection of influenza B virus comprise primers selected from the group consisting of:

Forward primers:

(SEQ ID NO: 15)

GACTCGATATCGAGTCAAATGCAmGATGGTCTCmAmGmCmTmA,

(SEQ ID NO: 16)

GACTCGATATCGAGTCAAATGCAmAATGGTCTCmAmGmCmTmA,

and

(SEQ ID NO: 17)

GACTCGATATCGAGTCAAATGCAmGATGGTTTCmAmGmCmTmA,

and

Reverse Primers:

(SEQ ID NO: 18)

GACTCGATATCGAGTCCTCCTTTmTCCCATTCCATmTmCmAmTmT,

(SEQ ID NO: 19)

GACTCGATATCGAGTCCTCCCTTmTCCCATTCCATmTmCmAmTmT,

and

(SEQ ID NO: 20)

GACTCGATATCGAGTCCTCCTTTmCCCCATTCCATmTmCmAmTmT;

or

wherein the forward and reverse primers for detection of bovine diarrhea virus comprise primers selected from the group consisting of:

Forward primers:

(SEQ ID NO: 22)

GACTCGATATCGAGTCGGCCCACmTGTATTGCTmAmCmTmGmAmAmA,

and

(SEQ ID NO: 23)

GACTCGATATCGAGTCGGCCCACmTGCACTGCTmAmCmTmAmAmAmA,

and

Reverse Primer:

(SEQ ID NO: 24)

GACTCGATATCGAGTCTGTGATCmAACTCCmAmTmGmTmGmCmC,

wherein mN indicates that the base N is a methoxy base;

b) a reverse transcriptase enzyme;

c) a nicking enzyme;

d) a strand-displacement polymerase; and

e) directions for use of the aforementioned primers and enzymes for amplifying a polynucleotide molecule.

Assignments (3)

Continuity (4)

References Cited (189)