Perelygina Ludmila, Patrusheva Irina, Manes Nina, Wildes Martin J, Krug Peter, Hilliard Julia K
Viral Immunology Center, Department of Biology, Georgia State University, P.O. Box 4118, Atlanta, GA 30302-4118, USA.
J Virol Methods. 2003 May;109(2):245-51. doi: 10.1016/s0166-0934(03)00078-8.
A TaqMan based real-time PCR assay was developed for rapid detection and quantitation of herpes B virus (Cercopithecine herpesvirus 1) in clinical samples. The assay utilizes B virus-specific primers and a probe to the non-conserved region of the gG gene to discriminate B virus from closely related alphaherpesviruses. Fifty copies of B virus DNA could be detected with 100% sensitivity with a wide range of quantitation spanning 6 logs. The assay was highly reproducible with intra- and inter-assay coefficients of variation of 0.6 and 2.4%, respectively. Clinical utility of the developed real-time PCR was evaluated by testing genomic DNA prepared from B virus clinical isolates (n=23) and human and monkey clinical specimens (n=62). This novel method was also compared with conventional cell culture with respect to sensitivity and specificity. TaqMan PCR assay was shown to be equally specific and more sensitive than culture method (culture vs. PCR sensitivity 50%) and was able to identify all B virus clinical isolates tested. Fast, reliable assessment of B virus DNA in infected cells and tissues makes real-time PCR assay a valuable tool for diagnosis and management of B virus infections.
开发了一种基于TaqMan的实时PCR检测方法，用于临床样本中B病毒（猕猴疱疹病毒1型）的快速检测和定量。该检测方法利用B病毒特异性引物和针对gG基因非保守区域的探针，以区分B病毒与密切相关的α疱疹病毒。在6个对数的广泛定量范围内，可100%灵敏地检测到50个拷贝的B病毒DNA。该检测方法具有高度可重复性，批内和批间变异系数分别为0.6%和2.4%。通过检测从B病毒临床分离株（n = 23）以及人和猴临床标本（n = 62）制备的基因组DNA，评估了所开发实时PCR的临床实用性。还将这种新方法与传统细胞培养方法在敏感性和特异性方面进行了比较。结果表明，TaqMan PCR检测方法具有同等特异性，且比培养方法更灵敏（培养法与PCR法的敏感性分别为50%），并且能够鉴定所有检测的B病毒临床分离株。对感染细胞和组织中的B病毒DNA进行快速、可靠的评估，使得实时PCR检测方法成为B病毒感染诊断和管理的宝贵工具。
