We have developed a quantitative PCDR assay that uses pan-dengue primers to detect dengue virus sequence.
Materials and methods
Primers and probe design
PCR primers and probe were synthesized by Eurogentec (Seraing, Belgium). Their sequences are shown in Table 1, where they bind in the NC_001475 sequence, and their relative positions in Figure 2A. Generic pan-dengue primers, which amplify all four dengue serotypes, were used with a dengue 3 serotype-specific probe. The pan-dengue primers used are longer than normal PCR primers, so that all four dengue serotypes could be amplified despite some sequence variation.
Preparation of DNA constructs for template DNA
A plasmid construct, pMA-T, containing a 395-nucleotide fragment of the DENV3 sequence was synthesized by GeneArt (Life Technologies, Paisley, UK); this includes nucleotides 10312–10707 from the published sequence (NC_001475), which is located in the 3′ untranslated region (3′ UTR) of the virus. The plasmid was digested with restriction enzymes, which have sites on either side of the DENV3 sequence, producing template DNA in a linear form. Tenfold dilutions of the digested plasmid were used in the quantitative assays spiked with 10 ng/µL of Aedes aegypti mosquito pupae RNA/DNA.
All amplification reactions were carried out on a T3000 Thermocycler (Biometra, Goettingen, Germany). Thermocycler conditions consisted of an initial denaturation 95°C for 2 min, and 34 cycles of 95°C for 5 s, 55°C or 15 s, 72°C for 15 s, and a final elongation of 72°C or 1 min. Eighteen microliters of the reaction were run on a 1.5% agarose gel and visualized by ethidium bromide staining. For sequencing of the amplicons, amplification product was separated by electrophoresis and gel purified, this was cloned into pJET1.2 (Fermentas, St. Leon-Rot, Germany) and transformed into XL-10 Gold cells (Stratagene, Santa Clara, CA, USA). Individual colonies were screened using primers that flank the cloning site, and the resulting PCR product was purified and sequenced by GATC (Konstanz, Germany).
Real-time amplification procedure
All qPCRs were performed using a Mx3005P (Stratagene) and analyzed with MxPro software (Stratagene). Assay components were optimized and consisted of 1× PCDR master mix (GeneFirst, Oxford, UK), 200 nM F2 primer, 60 nM F3 primer, 200 nM R1b primer, 100 nM R2 primer, and 200 nM Den3-Cy5 probe. Total reaction volume was 25 µL. The conditions consisted of an initial denaturation 95°C for 9 min and 45 cycles of 95°C for 6 s, 55°C for 30 s, and 72°C for 30 s. Assays using TaqMan Master Mix consisted of 1× TaqMan Master Mix (Applied Biosystems, Carlsbad, CA, USA), 200 nM F2 primer, 200 nM R1b primer, and 200 nM Den3-Cy5 probe. The conditions for qPCR using the TaqMan Master Mix consisted of an initial denaturation 95°C for 9 min and 45 cycles of 95°C for 6 s, 55°C for 30 s, 60°C for 20 s, and 72°C for 20 s. Reactions were carried out in clear plates and sealed with flat lid strips. All reactions were performed in triplicate or were done two or three times in separate experiments. Water was used for the no template control (NTC).
Intrathoracic injection of Aedes aegypti mosquitoes
Five-day-old adult, female Aedes aegypti RexD mosquitoes were intrathoracically inoculated with 4 × 102 pfu DENV3 (MX BC285/97). One week after inoculation, infection rates were confirmed by an immunofluorescence assay (IFA) on head squash smears using 5D4–11 MAb specific for DENV3. Mosquitoes were placed in RNAlater (Life Technologies) and stored at −20°C.
RNA extraction and cDNA synthesis
Total RNA was extracted from mosquitoes with TRIzol (Life Technologies) according to the manufacturer's instructions. Mosquitoes were homogenized using a plastic pestle. RNA was checked for quality by gel analysis and quantity by spectrometry using GeneQuant II (GE Healthcare, Little Chalfont, UK) and stored at -80°C. A maximum of 5 µg RNA was converted to cDNA using SuperScript II Reverse Transcriptase (Life Technologies) with a mixture of R1b and R2 as the first strand primer, according to the manufacturer's instructions. cDNA was treated with RNaseH (Life Technologies), stored at −20°C, and 1 µL cDNA was used in subsequent PCRs.
Results and discussion
Comparison of PCDR with PCR in gel-based assays
Conventional PCR only uses two primers in each reaction, in comparison to PCDR with multiple primers. To compare the sensitivity of PCR with PCDR, amplifications were carried out using four, three, or two of the pan-dengue primers with 140,000, 14,000, or 1400 copies of DENV3 template DNA, and the amplification products visualized by gel electrophoresis (Figure 2B). Only one amplification product was produced with two primers, but the use of three or four primers produced two or three PCR products, respectively, showing that amplicons were produced using all the primers available. In the reaction with 1400 copies of template DNA using two primers, very little PCR product was produced. However, using three or four primers in PCDR, there was a clear increase in the amount of product, with four primers being more sensitive than three. The amplification products were sequenced to confirm specificity (i.e., that using multiple primers in PCDR did not produce any off-target amplicons). Sequencing confirmed that only the specific products were amplified; furthermore, all four predicted PCDR products were indeed amplified (data not shown). Although Figure 2B shows some higher molecular weight products with PCDR (e.g., lane 2, ~450 bp), these were sequenced and found to contain only the nominal sequence and are therefore presumably oligomers of the predicted products.
Comparison of PCDR with PCR using PCDR enzyme for quantitative assays
To determine if PCDR can improve the sensitivity of quantitative assays, the DENV3 specific probe was used in combination with four, three, or two of the pan-dengue primers. Before doing this, we first tested if the single primer pairs in the four different combinations behaved differently. The result revealed that there was no significant difference in the quantification cycle (Cq) value or sensitivity for each primer pair (Table 2 and Supplementary Table S1). To compare PCDR and PCR, we chose the inner-most primer pair for the PCR. The sensitivity of each primer set was determined using 10-fold dilutions of DENV3 template DNA (Table 2 and Supplementary Table S1). Amplification curves for all dilutions are shown in Figure 3A, comparing four-primer PCDR with two-primer PCR; amplification curves of all primer sets for the reaction with 20 copies of templates are shown in Figure 3B. These results show that both lower Cq values and much improved fluorescence curves were achieved in four-primer PCDR in comparison to two-primer PCR for all dilutions tested. The Cq values were reduced by three cycles when using four primers instead of two. Efficiency was calculated by comparing Cq number against starting copy number and determining the slope of the graph produced. An efficiency of over 100% means a greater than 2-fold increase in amplicon per amplification cycle. The assays using four primers had an efficiency of around 107% (average from data of Tables 2 and 4); this is in comparison to 103% (average from data of Table 2 and Supplementary Table S1) when two primers were used. Although the total concentration of primer present was increased in the four-primer PCDR reaction, when the primer concentration was doubled in a two-primer PCR, there was no change in Cq or efficiency (data not shown), showing that the lower Cq value in PCDR was not simply due to an increase in total primer concentration. Although the slight increase of efficiency is not likely to be of practical importance in most contexts, the consistently lower Cq value would mean increased sensitivity and speed of reaction.