BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm.
BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm.
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DOI：
10.1186/1742-4690-7-91
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发表时间：
2010-11-02
期刊：
影响因子：
3.3
通讯作者：
Aida Y
中科院分区：
文献类型：
--
作者：
Jimba M;Takeshima SN;Matoba K;Endoh D;Aida Y
牛白血病病毒(BLV)与人T细胞白血病病毒(HTLV)关系密切，是地方性牛白血病的病原体，该病的特点是病程延长，常累及持续性淋巴细胞增多并最终导致B细胞淋巴瘤。即使在没有可检测到的BLV抗体的情况下，BLV前病毒仍然整合在细胞基因组中。因此，为了了解BLV诱发白血病的机制和进行BLV感染动物的筛选，需要对BLV感染牛整个病程中前病毒负荷的变化进行详细的评估。本研究的目的是建立一种新的定量实时聚合酶链式反应(PCR)方法，利用共同基序的协调(COCOMO)引物来检测临床动物中已知和新的BLV变异株的前病毒载量。利用专为检测多种病毒而开发的COCOMO算法，从GenBank中356个BLV序列中鉴定出52个BLV长末端重复序列，设计简并引物。从49个候选引物中筛选出最具特异性的72对引物，进行实时定量聚合酶链式反应后的熔融曲线分析，筛选出特异性最强的一组用于BLV LTR的检测。使用BLV TaqMan内部探针来提高检测的特异性和敏感性，并通过平行扩增单拷贝宿主基因(牛白细胞抗原DRA基因)来归一化基因组DNA。该方法具有高度的特异性、敏感性、定量和重复性，并能用先前建立的套式聚合酶链式反应方法在许多阴性样本中检测到BLV。该检测在检测来自国际各地的牛的BLV方面也是非常有效的。最后，这项试验使我们能够证明，前病毒载量不仅与合胞体形成评估的BLV感染能力有关，而且与BLV疾病进展相关。使用我们新开发的BLV-Cocomo-qPCR方法，我们能够检测到广泛的BLV突变病毒。Cocomo算法可能是一种有用的设计简并引物的工具，可用于定量其他逆转录病毒包括HTLV和人类免疫缺陷病毒1型的前病毒载量。
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals. Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.
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影响因子：
2.6
作者：
Kobayashi S;Tsutsui T;Yamamoto T;Hayama Y;Kameyama K;Konishi M;Murakami K
通讯作者：
Murakami K
影响因子：
3
作者：
Katoh K;Toh H
通讯作者：
Toh H
DOI：
10.1006/bbrc.1994.2444
发表时间：
1994-10-14
影响因子：
3.1
作者：
AIDA, Y;KOHDA, C;ASAHINA, M
通讯作者：
ASAHINA, M
DOI：
10.1073/pnas.77.5.2577
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
KETTMANN, R;CLEUTER, Y;CHANTRENNE, H
通讯作者：
CHANTRENNE, H
影响因子：
5.4
作者：
ADACHI, A;GENDELMAN, HE;MARTIN, MA
通讯作者：
MARTIN, MA