验证一种内部对照的一步法实时多重 RT-PCR 检测方法，用于检测和定量血浆中的登革病毒 RNA。
Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma.
机构信息
Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, Quan 5, Ho Chi Minh City, Viet Nam.
出版信息
J Virol Methods. 2011 Nov;177(2):168-73. doi: 10.1016/j.jviromet.2011.08.002. Epub 2011 Aug 5.
Abstract
Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
摘要
登革热是一种由蚊子传播的病毒感染，每年在全球范围内导致约 5000 万例临床明显病例。本研究开发了一种内部对照的一步实时多重 RT-PCR 检测法，使用特异性引物和荧光 TaqMan 探针，对血浆样本中的 DENV RNA 进行检测和定量。所有引物和探针均针对 NS5 基因 3'端附近的序列。该方法包括两个多重检测，对其灵敏度、特异性、线性、重复性和精密度进行了验证。在每份临床标本中加入内部对照模板，以保证每个实验步骤的质量。该检测方法可检测到 0.5 至 3 个感染性颗粒/毫升，快速且已在越南胡志明市热带病医院的两项治疗干预试验中的 287 例越南登革热患者中进行了操作特征描述。
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本文引用的文献
1
Kinetics of plasma viremia and soluble nonstructural protein 1 concentrations in dengue: differential effects according to serotype and immune status.
J Infect Dis. 2011 May 1;203(9):1292-300. doi: 10.1093/infdis/jir014. Epub 2011 Feb 18.
2
Kinetics of non-structural protein 1, IgM and IgG antibodies in dengue type 1 primary infection.
Virol J. 2011 Feb 2;8:47. doi: 10.1186/1743-422X-8-47.
3
Epidemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Vietnam.
Am J Trop Med Hyg. 2011 Jan;84(1):127-34. doi: 10.4269/ajtmh.2011.10-0476.
4
2nd International external quality control assessment for the molecular diagnosis of dengue infections.
PLoS Negl Trop Dis. 2010 Oct 5;4(10):e833. doi: 10.1371/journal.pntd.0000833.
5
The early whole-blood transcriptional signature of dengue virus and features associated with progression to dengue shock syndrome in Vietnamese children and young adults.
J Virol. 2010 Dec;84(24):12982-94. doi: 10.1128/JVI.01224-10. Epub 2010 Oct 13.
6
Multi-country evaluation of the sensitivity and specificity of two commercially-available NS1 ELISA assays for dengue diagnosis.
PLoS Negl Trop Dis. 2010 Aug 31;4(8):e811. doi: 10.1371/journal.pntd.0000811.
7
A randomized controlled trial of chloroquine for the treatment of dengue in Vietnamese adults.
PLoS Negl Trop Dis. 2010 Aug 10;4(8):e785. doi: 10.1371/journal.pntd.0000785.
8
Emergence of the Asian 1 genotype of dengue virus serotype 2 in viet nam: in vivo fitness advantage and lineage replacement in South-East Asia.
PLoS Negl Trop Dis. 2010 Jul 20;4(7):e757. doi: 10.1371/journal.pntd.0000757.
9
Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses.
BMC Infect Dis. 2010 May 28;10:142. doi: 10.1186/1471-2334-10-142.
10
Timing of CD8+ T cell responses in relation to commencement of capillary leakage in children with dengue.
J Immunol. 2010 Jun 15;184(12):7281-7. doi: 10.4049/jimmunol.0903262. Epub 2010 May 12.
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