High resolution melt genotyping of IBV, CSFV and NDV
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.
1. A method of characterizing one or more Newcastle Disease Virus (NDV) strains isolated from one or more animals comprising the steps of:
a) generating an NDV cDNA from the NDV strains isolated from the animals;
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 or M gene of NDV genome, and wherein the real-time PCR comprises the steps of:
i) initial denaturation at 95° C. for 3 seconds;
ii) denaturation at 95° C. for 1 minute;
iii) annealing at 60° C. for 1 minute;
iv) extension at 72° C. for 1 minute;
v) repeating steps ii)-iv) 45 times;
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 the HRM curve thereby characterizing the NDV strain, wherein the primer pair comprises 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 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;
wherein a saturating dye suitable for HRM is used.
2. The method of claim 1 , wherein the one or more NDV strains are selected from the group consisting of, Avinew strain, LaSota strain, MVP-Mukteswar strain, ND-B1 strain, Korea Dalguban strain, Herts 33 strain, Essex'70 strain, 135/93 strain, 617/83 strain, 34/90 strain, Beaudette C strain, D26 strain, MC110 strain, and 1154/98 strain.
3. The method of claim 1 further comprising the steps of sequencing the amplicon and identifying a novel strain of NDV wherein the F or M sequence of the NDV strain has less than 95% sequence identity to known NDV F or M sequences.
4. The method of claim 3 further comprising the step of isolating the novel strain of NDV.
5. The method of claim 1 , wherein the process further comprises the step of identifying whether the NDV strain is a vaccine-type strain or a wild-type strain that infected the animals.