一种用于研究伪狂犬病病毒潜伏感染的基于荧光的定量PCR方法。

A fluorescence-based quantitative PCR method for investigation of pseudorabies virus latency.

作者信息

Thiery R, Pannetier C, Rziha H J, Jestin A

机构信息

Centre National d'Etudes Vétérinaires et Alimentaires, Laboratoire de Biologie Moléculaire, Ploufragan, France.

出版信息

J Virol Methods. 1996 Sep;61(1-2):79-87. doi: 10.1016/0166-0934(96)02072-1.

DOI:10.1016/0166-0934(96)02072-1

PMID:8882940

Abstract

A quantitative PCR method was developed in order to quantitate the number of copies of Pseudorabies virus (PRV) genome present in tissues from infected pigs. The method is based on the use of an internal standard that differs from the target DNA by a deletion of ten base pairs, and that is co-amplified with the target DNA. The resulting PCR products are labelled with a fluorescent primer and are then separated and detected by means of an automated sequencer. The assay was found to be specific and sensitive, allowing the detection of five copies of viral DNA among 10(6) host cells. The method was used successfully to quantitate the number of PRV DNA copies in trigeminal ganglia samples from infected pigs during the acute and the latent stages of the infection. Between 12 and 3.10(5) copies of viral genome per 10(6) neuronal cells were detected in these tissues which is consistent with data published previously.

摘要

为了定量感染猪组织中伪狂犬病病毒（PRV）基因组的拷贝数，开发了一种定量PCR方法。该方法基于使用一种内标，该内标与靶DNA的区别在于缺失了10个碱基对，并且与靶DNA共同扩增。所得的PCR产物用荧光引物标记，然后通过自动测序仪进行分离和检测。该检测方法具有特异性和灵敏性，能够在10⁶个宿主细胞中检测到5个病毒DNA拷贝。该方法成功用于定量感染猪在感染急性期和潜伏期三叉神经节样本中PRV DNA的拷贝数。在这些组织中，每10⁶个神经元细胞中检测到12至3×⁵个病毒基因组拷贝，这与先前发表的数据一致。