Method and kit for the field diagnosis of caprine arthritis-encephalitis virus (CAEV) infection

Patent metadata

Field	Value
Publication number	US-10161012-B2
Application number	US-201715439359-A
Country	US
Kind code	B2
Filing date	Feb 22, 2017
Priority date	Feb 22, 2017
Publication date	Dec 25, 2018
Grant date	Dec 25, 2018

Abstract

The invention is to provide a method and kit based on recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) for detection of caprine arthritis-encephalitis virus (CAEV) infection. The method and kit are suitable for both laboratory and field application, and are specific and sensitive for detecting CAEV proviral DNA in goats in a fast manner. The method and lit of the invention are also applicable for on-site utilization at farms and should be useful in both eradication programs and epidemiological studies.

First claim

What is claimed is:

1. A method for detection of caprine arthritis-encephalitis virus (CAEV) infection, comprising the steps of:
(a) amplifying gag-segment (nt 618-803) DNA of CAEV as a target sequence in the sample by recombinase polymerase amplification (RPA) in the presence of a pair forward and reverse primers and a probe sequence which is complementary to an internal region of the target sequence, wherein one of the primers being labeled with a first label and the probe sequence being labeled with a second label such that amplification of the target sequence generates an amplicon labeled with both first and second labels;
(b) applying an amount of the amplification product of step (a) to a lateral flow device/strip to flow laterally towards a distal end of the lateral flow device/strip, wherein the lateral flow device/strip sequentially comprising (i) a sample pad, (ii) a conjugate pad, (iii) a test region, and (iv) a control region, said conjugate pad having mobile reporter labeled with a first agent which specifically binds to the first label or the second label of the amplicon, said test region provided with a second agent which specifically binds to the second label or the first label of the amplicon to inhibit further lateral flow of the amplicon associated with the mobile reporter, and said control region being provided with a control agent; and
(c) detecting any binding of the amplification product at the test region and the control region.

2. The method of claim 1 , wherein the first and second labels are selected from biotin, fluorescein derivatives, rhodamine derivatives, Cascade Blue, Lucifer yellow, 5-bromo-2-deoxyuridine (BrdU), dinitrophenol (DNP), digoxygenin (DIG), and short peptide label sequences.

3. The method of claim 1 , wherein the forward primer comprises a sequence of SEQ ID NO: 1 and the reverse primer comprises a sequence of SEQ ID NO: 2.

4. The method of claim 3 , wherein the reverse primer comprises a 5′-biotin label.

5. The method of claim 3 , wherein the probe sequence consists of SEQ ID NO: 3 with 5′-FAM antigenic label and a downstream SEQ ID NO: 4 carrying a C3-spacer at its 3′ end, wherein SEQ ID NO: 3 and SEQ ID NO: 4 is connected by a THF spacer.

6. The method of claim 4 , wherein the mobile reporter is a microparticle of gold, silica, selenium, polystyrene, melamine resin, polymethacrylate, styrene/divinylbenzene copolymer or polyvinyltoluene.

7. A kit for detection of caprine arthritis-encephalitis virus (CAEV) in a sample, comprising:
a pair of forward and reverse primers targeting a gag-segment (nt 618-803) DNA of CAEV as a target sequence;
a probe sequence which is complementary to an internal region of the target sequence;
a reagent for conducting recombinase polymerase amplification (RPA) reaction; and
a lateral flow device/strip;
wherein one of the primers being labeled with a first label and the probe sequence being labeled with a second label such that amplification of the target sequence generates an amplicon labeled with both first and second labels; and the lateral flow device/strip sequentially comprising (i) a sample pad, (ii) a conjugate pad, (iii) a test region, and (iv) a control region, said conjugate pad having mobile reporter labeled with a first agent which specifically binds to the first label or the second label of the amplicon, said test region provided with a second agent which specifically binds to the second label or the first label and said control region being provided with a control agent.

8. The kit of claim 7 , which further comprises a reagent for extracting nucleic acid from the sample.

9. The kit of claim 8 , wherein the forward primer comprises sequence of SEQ ID NO: 1 and the reverse primer comprises a sequence of SEQ ID NO: 2.

10. The kit of claim 9 , wherein the reverse primer comprises a 5′-biotin label.

11. The kit of claim 10 , wherein the probe sequence consists of SEQ ID NO: 3 with 5′-FAM antigenic label and a downstream SEQ ID NO: 4 carrying a C3-spacer at its 3′ end, wherein SEQ ID NO: 3 and SEQ ID NO: 4 is connected by a THF spacer.

12. The kit of claim 11 , wherein the mobile reporter is a microparticle labeled with an anti-FAM antibody.

13. The kit of claim 12 , wherein the control agent is a secondary antibody of the anti-FAM antibody.

Assignees

• Univ Nat Taiwan

Inventors

• Wang Pei-Hwa
• Tu Po-An
• Lai Fang-Yu
• Shiau Jen-Wen

Classifications

• G01N33/558

  using diffusion or migration of antigen or antibody {(immunochromatographic test strips G01N33/54387)} · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/702 Primary

  for retroviruses · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/682

  Signal amplification · CPC title