Assay methods for MDV-1

摘要

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 is of vaccine and virulent viruses.

主权项

1. A method for the quantification of a vaccine strain and 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; (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 said SNP being a G/A polymorphism located at position 320 of the sequences of FIG. 1 ((a) and (b)); and(b) contacting the amplified nucleic acid of (a) with a detectable nucleic acid probe specific for the SNP of the vaccine strain and a detectable nucleic acid probe specific for the SNP of the virulent strain of MDV-1; wherein the detectable nucleic acid probes comprise 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) (iii) measuring the detectable signal produced by each of the vaccine probe arid the virulent strain probe of (ii); and (iv) comparing the detectable signal measured in (iii) for each specific probe to determine the amount of virulent and vaccine strain in the sample.