Development of a real-time PCR assay using SYBR Green chemistry for monitoring Marek's disease virus genome load in feather tips

被引:77
作者
Abdul-Careem, Mohamed Faizal
Hunter, Bruce D.
Nagy, Eva
Read, Leah R.
Sanei, Babak
Spencer, J. Lloyd
Sharif, Shayan [1 ]
机构
[1] Univ Guelph, Ontario Vet Coll, Dept Pathobiol, Guelph, ON N1G 2W1, Canada
[2] Ontario Minist Agr & Food, Guelph, ON N1G 4Y2, Canada
[3] Canadian Food Inspect Agcy, Ottawa, ON K2E 8A5, Canada
基金
加拿大自然科学与工程研究理事会; 加拿大创新基金会;
关键词
real-time PCR; Marek's disease virus; feather follicle;
D O I
10.1016/j.jviromet.2005.10.018
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Feather follicles of birds infected with Marck's disease virus (MDV) serve as the sole source of infectious virus particles. The present study was aimed at developing a SYBR Green real-time PCR assay to detect and quantify MDV loads in feather tips targeting meq gene of the virus. The assay had a dynamic range of 8 logs, mean inter- and intra-assay coefficient variation (CV) of < 5% and minimum detection limit of 15 MDV genome copies when plasmid DNA was used as the template. The sensitivity of the assay was compared with that of the conventional PCR technique and found to be 2.5-10 times more sensitive than the conventional PCR technique. The assay was validated using feather tip DNA preparations derived from chickens infected with 250 plaque forming units (PFU) of RB1B strain of MDV and sampled on days 7, 14, 21 and 28 post-infection (p.i.) along with uninfected chickens. MDV genome was quantifiable in feather tips of infected birds by day 7 p.i. and the number of MDV copies peaked by day 14 p.i., but then gradually decreased by day 28. This reliable real-time PCR assay may be used for monitoring MDV genome loads in tissues of experimentally or naturally infected birds. (c) 2005 Elsevier B.V. All rights reserved.
引用
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页码:34 / 40
页数:7
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