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. 2005 Oct;51(10):1885-8.
doi: 10.1373/clinchem.2005.054106.

Use of dual TaqMan probes to increase the sensitivity of 1-step quantitative reverse transcription-PCR: application to the detection of SARS coronavirus

Shea Ping Yip  1 , Shing Shun T To, Polly H M Leung, Tsz Shan Cheung, Peter K C Cheng, Wilina W L Lim
Affiliations

Use of dual TaqMan probes to increase the sensitivity of 1-step quantitative reverse transcription-PCR: application to the detection of SARS coronavirus

Shea Ping Yip et al. Clin Chem. 2005 Oct.
No abstract available

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Figures

Figure 1.
Figure 1.
RT-PCR assay design and performance. (A), 1-step RT-PCR assays with 1 (assay P1) or 2 TaqMan probes (assays P2a to P2c). The length of each amplicon is indicated in parentheses. The same forward primer was used for the 3 dual-probe assays and was upstream of that for the 1-probe assay. The arrowheads indicate the 3′ end of a primer or probe. (B), names and sequences of the primers and the TaqMan MGB probes shown together with the symbols used in A. Note that the TaqMan MGB probes (Applied Biosystems) are labeled with a 5′ reporter dye, 6-carboxyfluorescein (FAM), and a 3′ nonfluorescent quencher (NFQ) plus a minor grove binder (MGB) that stabilizes the probe–target duplex by binding the minor groove of double-stranded DNA (31). (C), amplification plot of FAM fluorescence intensity against the PCR cycle for the P1 one-probe assay. Delta Rn (y axis) indicates the magnitude of the signal intensity generated by a given set of PCR conditions and is obtained from the equation: delta Rn = (Rn+) − (Rn−). The Rn+ value is obtained as a ratio of FAM fluorescence intensity to the fluorescence intensity of the passive reference dye (ROX) included in the reaction mixture for a PCR with template. The Rn− value is similarly obtained as a ratio for a PCR without template (the no-template control). The RNA copy numbers per reaction are indicated on the right for each curve. (D), amplification plot of FAM fluorescence intensity against the PCR cycle for the P2a dual-probe assay. Assays P2b and P2c produced similar amplification plots (data not shown). (E), calibration curves for the P1 one-probe assay and the P2a dual-probe assay. Assays P2b and P2c produced calibration curves very similar to that for assay P2a (data not shown). (F), comparison of the RNA copy number per mL of input RNA sample determined by Artus assay (x axis) and the P2a dual-probe assay (y axis). The RNA samples were extracted from 18 confirmed SARS cases with 6 cases each providing stool, nasopharyngeal aspirate, and serum specimens.
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