Establishment of Real-Time PCR Method for Detection of Extraneous Marek’s Disease Virus

In order to solve the problems of low sensitivity, long detection time, and poor discrimination of existing exogenous Marek’s disease virus (MDV) testing methods, this study was designed to establish two real-time PCR detection methods for the identification of MDV serotype 1 (MDV 1) and MDV serotype 3 (MDV 3) strains, which could be used for purity control of poultry-derived biological products.

The UL19 sequences of MDV 1, MDV serotype 2 (MDV 2) and MDV 3 strains were downloaded from the NCBI database and were used for nucleotide and amino acid homology comparison. A pair of specific primers and corresponding Taqman probe was designed from the known sequence of conserved UL19 of MDV 1 CVI988 strain and MDV 3 FC126 strain, respectively, and two real-time PCR detection methods were established. The corresponding recombinant plasmids were constructed and used as positive standards to make standard curves, and the sensitivity of gene copy number of the methods were evaluated. Other avian virus-associated biological products, virus, the full-length plasmid of UL19 of MDV 2 SB-1 strain and the raw materials for production (SPF chicken embryo allantoic fluid, embryonic body, allantoic membrane, chicken embryo fibroblasts) were detected to evaluate the specificity of the established methods. 600, 60, 6, 0.6, 0.06, 0.006 and 0.0006 PFU of CVI988 or FC126 strains were detected, respectively, and the sensitivity of the established two methods for detecting live virions was evaluated. Three repeatability tests were performed using corresponding recombinant plasmids of different dilutions, and the correlation coefficient were calculated to analyze the reproducibility of the two established detection methods.

The nucleotide and the derived amino acid homology of MDV UL19 in the same serotype was highly conserved with 99.99%, and the nucleotide homology between different serotypes was only about 75%, while the derived amino acid homology was only about 85%. MDV 1 and MDV 3 real-time PCR detection methods were established, respectively. About the MDV 1 real-time PCR detection method, the amplification efficiency was 98.8%, the correlation coefficient was 0.992, with the standard curve: Y=-3.351X+38.828 (Y = Ct, X = lg ( copy number)). About the MDV 3 real-time PCR detection method, the amplification efficiency was 95%, the correlation coefficient was 0.998, and the standard curve: Y=-3.447X+36.496 (Y = Ct, X = lg ( copy number)). The established detection methods could specially detect MDV 1 or MDV 3 without detecting any other avian virus-associated biological products, virus, the full-length plasmid of UL19 of MDV 2 SB-1 strain, along with production materials for poultry. The sensitivity of MDV 1 real-time PCR detection method was high, with the gene copy number detection limit of 32.8 copies/μL, which could detect at least 0.006 PFU of CVI988 strain. The sensitivity of MDV 3 real-time PCR detection method was high, with the gene copy number detection limit of 10 copies/μL, which could detect at least 0.006 PFU of FC126 strain. The coefficient of variation of the repeatability test was less than 1% in MDV 1 real-time PCR detection method, and less than 1.5% in MDV 3 real-time PCR detection method, respectively.

The established real-time PCR detection methods would be beneficial for detecting exogenous MDV 1 and MDV 3 strains in poultry-derived biological products.