Assay methods for MDV-1
View Patent ↗A method for the quantification of a vaccine strain and/or a virulent strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, comprising the steps of: (i) providing a biological sample from the bird and optionally isolating nucleic acid from the biological sample; (ii) subjecting the biological sample of (i) to real-time quantitative PCR (qPCR) comprising: (a) amplification of a region of the pp38 gene within the nucleic acid sample of (i), said region containing a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1; and (b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the vaccine strain of MDV-1 and/or a detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1; and (iii) Measuring changes in the detectable signal produced by the probe of (ii). Methods are also provided for the absolute quantification of vaccine and virulent viruses.
1. A kit of parts for the discrimination between and quantification of a vaccine strain and a virulent strain of Marek's Disease Virus Serotype-1 (MDV-1) in a sample from a bird, the kit comprising:
(i) primers that will amplify a region of the pp38 gene incorporating a consistent single nucleotide polymorphism (SNP) difference between vaccine and virulent strains of MDV-1 said SNP being a G/A polymorphism located at position 320 of the sequences of FIG. 1 ((a) and (b));
(ii) a detectable nucleic acid probe specific for the SNP of the vaccine strain of MDV-1; and
(iii) at least one detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1, wherein the specific nucleic acid probes of (ii) and (iii) are each labelled with at least one signal-generating dye and wherein the probes compromise the following sequences:
vaccine strain specific probe sequence: 5′ CCCACCGTGACAGCC 3′ (SEQ ID NO: 1); and at least one of the following virulent strain-specific probe sequences: 5′ CCCACTGTGACAGCC 3′ (SEQ ID NO: 2) and 5′ CTCCCACTGTGACAGCC 3′ (SEQ ID NO: 3).
2. The kit of claim 1 wherein the kit further comprises standard qPCR reagents.
3. The kit of claim 1 further comprising reagents required for a known standard qPCR utilizing a probe labelled with a different signal-generating dye than that of each of the specific nucleic acid probes of (ii) and (iii) to allow absolute quantification of the vaccine and virulent strains.
4. The kit of claim 1 wherein the vaccine strain is the CVI1988 vaccine.
5. The kit of claim 1 wherein the kit further comprises a total probe that binds both the vaccine strain and the virulent strain.
6. The kit of claim 5 wherein the total probe comprises the following sequence:
(SEQ ID NO: 4)
5′ GCTACCGCCTGAGCC 3′.
7. The kit of claim 1 wherein the primers are:
Forward primer:
(SEQ ID NO: 5)
5′ GAGCTAACCGGAGAGGGAGA 3′;
and
Reverse primer:
(SEQ ID NO: 6)
5′ CGCATACCGACTTTCGTCAA 3′.
8. The kit of claim 1 wherein the virulent MDV strain is selected from RB1B MDV, 584A MDV, 595 MDV, 684A MDV, Md5 MDV, HPRS-B14 MDV, JM102 MDV, 660A MDV, 675A MDV, 549 MDV, 571 MDV, or C12-130 MDV.