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doi: 10.1155/2011/514681. Epub 2011 Dec 5.

Internally controlled, generic real-time PCR for quantification and multiplex real-time PCR with serotype-specific probes for serotyping of dengue virus infections

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Internally controlled, generic real-time PCR for quantification and multiplex real-time PCR with serotype-specific probes for serotyping of dengue virus infections

Sandra Menting et al. Adv Virol. 2011.

Abstract

Dengue has become a global public health problem and a sensitive diagnostic test for early phase detection can be life saving. An internally controlled, generic real-time PCR was developed and validated by testing serial dilutions of a DENV positive control RNA in the presence of a fixed amount of IC with results showing a good linearity (R(2) = 0.9967) and a LOD of at least 1.95 ×ばつ 10(4) copies/mL. Application of the generic PCR on 136 patient samples revealed a sensitivity of 95.8% and specificity of 100%. A newly developed multiplex real-time PCR with serotype-specific probes allowed the serotyping of DENV for 80 out of 92 (87%) generic real-time PCR positive patients. Combined these real-time PCRs offer a convenient diagnostic tool for the sensitive and specific quantification of DENV in clinical specimens with the possibility for serotyping.

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Figures

Figure 1
Figure 1
The dynamic range of generic of PCR. Mean Cq-values of twelve replicates per each 10-fold dilutions of DENV in vitro RNA extracted with a fixed amount of 6.25 ×ばつ 103 IC copies in extraction in the back ground of DENV negative serum and tested in generic real-time PCR resulting in a dynamic range of 6.25 ×ばつ 103–6.25 ×ばつ 107 copies/mL of DENV in vitro RNA with a regression coefficient of 0.9967.
Figure 2
Figure 2
Multiple sequence alignments of DENV 3′UTR region deduced from complete genome sequences of dengue serotypes 1–4 (DENV 1–4) obtained from GenBank showing primers and probes sequences used in generic and/or multiplex real-time PCR with serotype-specific probes for detection of DENV in vitro RNA and DENV in patient samples.

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