Development of an Internally Controlled Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Comparison of Four Molecular Dengue Virus Detection Assays

被引:41
作者
Waggoner, Jesse J. [1 ]
Abeynayake, Janaki [2 ]
Sahoo, Malaya K. [2 ]
Gresh, Lionel [3 ]
Tellez, Yolanda [4 ]
Gonzalez, Karla [4 ]
Ballesteros, Gabriela [4 ]
Balmaseda, Angel [4 ]
Karunaratne, Kumudu [5 ]
Harris, Eva [6 ]
Pinsky, Benjamin A. [1 ,2 ]
机构
[1] Stanford Univ, Sch Med, Dept Med, Div Infect Dis & Geog Med, Stanford, CA 94305 USA
[2] Stanford Univ, Sch Med, Dept Pathol, Stanford, CA 94305 USA
[3] Sustainable Sci Inst, Managua, Nicaragua
[4] Minist Hlth, Ctr Nacl Diagnost & Referencia, Natl Virol Lab, Managua, Nicaragua
[5] Lady Ridgeway Hosp, Dept Med Microbiol, Colombo, Sri Lanka
[6] Univ Calif Berkeley, Sch Publ Hlth, Div Infect Dis & Vaccinol, Berkeley, CA 94720 USA
基金
美国国家卫生研究院;
关键词
PRIMARY-SCHOOL CHILDREN; RAPID DETECTION; HEMORRHAGIC-FEVER; ANTIBODY-RESPONSE; KAMPHAENG PHET; RT-PCR; SEROTYPE; DISEASE; INFECTION; QUANTIFICATION;
D O I
10.1128/JCM.00548-13
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log(10) cDNA equivalents/mu l and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/mu l, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P <= 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided.
引用
收藏
页码:2172 / 2181
页数:10
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