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Assay methods for detection of a virus in an avian tissue sample

A technology for tissue samples and viruses, applied in the determination/examination of microorganisms, biochemical equipment and methods, etc., can solve the problems of cumbersome routine diagnosis and so on

Inactive Publication Date: 2005-12-07
WYETH LLC
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  • Abstract
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  • Application Information

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Problems solved by technology

In situ hybridization has been used to detect the MDV genome in infected tissues (Endoh et al (1996) Journal of Veterinary Medical Science, 58, 969-976; Ross et al (1997) Journal of General Virology, 78, 2191-2198) , but the method is too cumbersome for routine diagnosis

Method used

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  • Assay methods for detection of a virus in an avian tissue sample
  • Assay methods for detection of a virus in an avian tissue sample
  • Assay methods for detection of a virus in an avian tissue sample

Examples

Experimental program
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Embodiment 1

[0079] Example 1: Quantitative PCR assay of MDV-1

[0080] Taqman TM Quantitative PCR is an established technique for quantifying the starting number of PCR target sequences by determining the number of PCR cycles required to reach a fluorescence threshold (determined mathematically from the Ct value). The higher the copy number of the target sequence in the sample, the fewer PCR cycles are required to reach the Ct threshold.

[0081] Taqman primers and probes were designed according to the Meq gene sequence of MDV strain GA (see below). This sequence was published in: Jones D, Lee L, Liu JL, Kung HJ, Tillotson JK. Marek disease virus encodes a basic-leucine zipper gene resembling the fos / junoncogenes that is highly expressed in lymphoblastoma Lymphoblastoid tumors.) Proc Natl Acad Sci USA. 1992 May 1; 89 (9): 4042-6. The sequence accession number is: M89471 (SEQ ID No. 9) using "Primer Express" ('Primer Express') of Applied Biosystems software to select the best primer / pr...

Embodiment 2

[0183] Example 2: Method for specifically detecting MDV-1 (CV1 988 vaccine) strains

[0184] This embodiment of the invention involves the use of a specially modified version of the 132 base pair (bp) repeat polymerase chain reaction (PCR) assay to detect the presence of CVI988 Marek's disease vaccine in chicken feathers.

[0185]The 132 base pair repetitive gene sequence is located within the repeat long (IR1) segment of the Marek's disease virus (serotype 1) genome. The complete genome sequence of MDV 1 was described by Tulman et al. (Sept 2000) J Virol. Vol. 74 No. 17, p7980-7988 and deposited in GenBank Accession No. AF243438. The CV1 988 isolate of serotype 1 Marek's disease (the vaccine strain) has multiple copies of the repeat, although the wild strain has only a single copy (Silva et al (1992) Avian Dis 36:521-528; and Becker et al (1993 ) Virus Genes 7:277-287). Measuring copy number offers the possibility to distinguish vaccine strains from wild strains (Becker et ...

Embodiment 3

[0274] Example 3 - Detection of the Rispens virus gene in feather tip DNA using PCR: comparison of samples from different feather regions.

[0275] method

[0276] Feather samples were collected from Rispens-vaccinated chicks

[0277] Two-week-old Rock Island Red chicks were injected intraperitoneally with 1000 pfu of Fort Dodge Rispens vaccine virus. Place age-matched unvaccinated chicks in a separate room. The wings of all chicks were banded to allow identification of individual chicks throughout the experiment. Samples were collected from 5 vaccinated chicks (#921, #923, #924, #925) and 1 non-vaccinated chick (#946) on days 8, 13, 19 and 26 post-vaccination. Approximately 6 feathers were plucked from each of the neck, shoulder, spine, underarm and femoral regions. The remaining 5 feather areas were not used, either because the number of feathers was too small to allow sampling at 4 points, or because it was considered inhumane to pluck the delicate skin areas of live ch...

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Abstract

The invention relates to methods of detecting a virus in an avian tissue sample wherein genetic material derived from feathers is tested for the presence of genetic material from the virus.

Description

[0001] The present invention relates to assay methods, in particular to assay methods for detecting the presence of viruses, in particular Marek's Disease Virus (MDV), in avian tissue samples. Background of the invention [0002] Marek's disease virus (MDV) is a herpes virus that causes lymphoproliferative disease in chickens. Even after the introduction of vaccines against MDV, infection still caused considerable losses to the chicken industry. MDV is divided into three serotypes, all of which produce latent infection. Serotype 1 includes oncogenic viruses, serotype 2 includes non-oncogenic viruses, and serotype 3 includes turkey herpesvirus (HVT) (Bulow et al (1976) Zentralblatt fur Veterinarmedizin, 23B, 391-402). [0003] The traditional diagnosis of Marek's disease is based on clinical signs and pathological changes. However, it is desirable to have more specific methods for monitoring the prevalence of MDV. Haider et al. describe the detection of viral antigens in fea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor V·K·奈尔S·J·拜根特R·J·W·库里
Owner WYETH LLC
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