High Resolution Melt Genotyping of IBV, CSFV and NDV
AssigneesAsia-Pacific Special Nutrients Sdn. Bhd., Merial Limited
InventorsJean-Christophe Audonnet, Seetha Jaganathan
Life of this patent
Estimating the term…
Abstract
The present invention relates to methods of differentiating and characterizing IBV, CSFV and NDV strains, and identifying new strains using high resolution melt technology. The present invention also provides primers and kits for use with such methods.
Claims30
A process or method of characterizing a strain of IBV comprising the steps of:
- a) generating an IBV cDNA from the IBV strain;
- b) exposing the cDNA to a primer pair comprising a forward primer and a reverse primer in a real-time polymerase chain reaction (PCR) to yield an amplicon, wherein the primer pair is specific to the S1 gene of IBV genome;
- c) performing high resolution melt (HRM) curve analysis on a double-stranded product comprising the amplicon immediately after the real-time PCR;
- and
- d) analyzing and comparing the HRM curve thereby characterizing the IBV strain.
A kit for detecting an IBV strain comprising:
- a) a primer pair comprising a forward primer having the sequence as set forth in SEQ ID NO:29 and a reverse primer having the sequence as set forth in SEQ ID NO:30;
- b) an instruction describing the parameters and conditions to perform real-time PCR.
An isolated polynucleotide or primer for HRM analysis having the sequence as set forth in SEQ ID NO:29 or SEQ ID NO:30.
A process or method of characterizing a strain of CSFV comprising the steps of:
- a) generating a CSFV cDNA from the CSFV strain;
- b) exposing the cDNA to a primer pair comprising a forward primer and a reverse primer in a real-time polymerase chain reaction (PCR) to yield an amplicon, wherein the primer pair is specific to the NS5B or 3′NTR region of CSFV genome;
- c) performing high resolution melt (HRM) curve analysis on a double-stranded product comprising the amplicon immediately after the real-time PCR;
- and
- d) analyzing and comparing the HRM curve thereby characterizing the CSFV strain.
A kit for detecting an CSFV strain comprising:
- a) A primer pair comprising a forward primer having the sequence as set forth in SEQ ID NO:8 and a reverse primer having the sequence as set forth in SEQ ID NO:9;
- b) an instruction describing the parameters and conditions to perform real-time PCR.
An isolated polynucleotide or primer for HRM analysis having the sequence as set forth in SEQ ID NO:8 or SEQ ID NO:9.
A process or method of characterizing a strain of NDV comprising the steps of:
- a) generating an NDV cDNA from the IBV strain;
- b) exposing the cDNA to a primer pair comprising a forward primer and a reverse primer in a real-time polymerase chain reaction (PCR) to yield an amplicon, wherein the primer pair is specific to the F, NP, P, M, FIN or L gene of NDV genome;
- c) performing high resolution melt (HRM) curve analysis on a double-stranded product comprising the amplicon immediately after the real-time PCR;
- and
- d) analyzing and comparing the HRM curve thereby characterizing the NDV strain.
A kit for detecting an NDV strain comprising:
- a) a primer pair comprising a forward primer having the sequence as set forth in SEQ ID NO:17 and a reverse primer having the sequence as set forth in SEQ ID NO:18;
- or a primer pair comprising a forward primer having the sequence as set forth in SEQ ID NO:31 and a reverse primer having the sequence as set forth in SEQ ID NO:32;
- and
- b) an instruction describing the parameters and conditions to perform real-time PCR.
An isolated polynucleotide or primer for HRM analysis having the sequence as set forth in SEQ ID NO:17, 18, 31, or 32.
Dependencies are inferred from the claim text.