Clinical Chemistry 53: 594-599, 2007.
First published February 22, 2007; 10.1373/clinchem.2006.077446
(Clinical Chemistry. 2007;53:594-599.)
© 2007 American Association for Clinical Chemistry, Inc.
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Molecular Diagnostics and Genetics |
One-Step Rapid Reverse Transcription–PCR Assay for Detecting and Typing Dengue Viruses with GC Tail and Induced Fluorescence Resonance Energy Transfer Techniques for Melting Temperature and Color Multiplexing
Constance L.H. Lo1,
Shea Ping Yip1,
Peter K.C. Cheng2,
Tony S.S. To1,
Wilina W.L. Lim2 and
Polly H.M. Leung1,a
1 Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China.
2 Public Health Laboratory Centre, Centre for Health Protection, Department of Health, Hong Kong SAR, China.
aAddress correspondence to this author at: Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China. Fax 852-2362-4365; e-mail htpolly{at}inet.polyu.edu.hk.
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Abstract
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Background: Dengue fever is an arthropod-borne infection caused by dengue viruses (DVs; DEN-1 to DEN-4). Early diagnosis is critical to prevent severe disease progression and the spreading of DV because no vaccine or specific treatment is available; therefore, a rapid and specific diagnostic assay capable of detecting and typing all serotypes would be ideal.
Methods: We amplified RNA samples from all 4 DV serotypes and Japanese encephalitis virus with 4 serotype-specific forward primers and a universal species-specific reverse primer. DEN-1 and DEN-3 forward primers were labeled at their 5' ends with BODIPY 630/650 and Cy5.5, respectively. DEN-1 and DEN-3 amplicons were detected by their characteristic emission generated from induced fluorescence resonance energy transfer. The presence of DEN-2 and DEN-4 amplicons was indicated by SYBR Green I (SGI) signals at specific amplicon melting temperatures (Tms).
Results: Fluorescence signals with specific emission wavelengths were obtained from DEN-1 and DEN-3. SGI melting profiles showed a Tm difference between DEN-2 and DEN-4 of 4.7 °C, which was sufficient for differentiating these 2 serotypes. The primers did not amplify the Japanese encephalitis virus. The detection limits of DEN-1 to DEN-4 were 1.64 x 10–4, 1.05 x 10–3, 8.15 x 10–4, and 5.80 x 10–3 plaque-forming units per reaction, respectively. The assay had a dynamic range of 103–108 plaque-forming units/L and could be performed in 2 h.
Conclusions: A single-tube, 1-step reverse transcription–PCR assay based on Tm and color multiplexing was developed for detecting and typing all 4 DV serotypes.
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Introduction
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Dengue fever is caused by dengue virus (DV)1
serotypes 1–4 (DEN-1 to DEN-4) and transmitted by infected female mosquitoes (Aedes aegypti and Aedes albopictus) (1). More than 50 million cases of dengue fever and dengue hemorrhagic fever are reported annually worldwide (2). The infection became epidemic (>700 000 cases) in Southeast Asia between 2001 and 2005. In tropical and subtropical countries, 52% of the population is at risk for the infection (3). Asymptomatic or flu-like symptomatic dengue fever may delay diagnosis, and the disease may progress to severe dengue hemorrhagic fever and lethal dengue shock syndrome (4)(5). Because vaccination and selective treatment for dengue infection are not available, rapid and specific diagnosis during early onset of the disease is critical and can help prevent progression of the disease through monitoring and supportive therapy (5)(6). Most importantly, early diagnosis can help prevent the spread of DV via mosquito bites from infected patients during the viremic phase.
Reverse transcription (RT)–PCR analysis has been used in the diagnosis of flavivirus infections (6)(7). DV shares genomic homology with other flaviviruses, and genotypic variations occur within the same serotype (8)(9). Thus, species- and serotype-specific detection of DV in the same tube is difficult. Currently, TaqMan assays (Applied Biosystems) are widely used for dengue detection (10)(11)(12)(13)(14)(15). Most of these assays amplify the 4 serotypes in 4 separate tubes (10)(11). Only recently has a 1-tube fourplex TaqMan assay been developed (13); however, the 4 pairs of primers and the 4 distinct fluorophores for dual-labeled probes increase the complexity and cost of the assay. Moreover, probe design is less flexible because a perfect match with the target sequence is required. The LightCycler approach (Roche Diagnostics), another platform with high diagnostic value, allows more efficient cycling for rapid detection of DVs (16). The detection strategies provided by the LightCycler methods facilitate multiplexing of melting temperature (Tm; temperature at which 50% of double-stranded DNA is denatured) and color in the assay (16). In this study, we aimed to develop a single-step LightCycler assay capable of detecting and typing DVs.
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Materials and Methods
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assay design
We applied 2 strategies offered by the LightCycler for developing a 1-step assay for detecting and typing DVs. The 1st strategy was to increase the Tm of an amplicon by means of a primer tagged with a GC tail (5'-CGC GCC GGC CGC GG-3'), which allowed distinguishing between DEN-2 and DEN-4 in channel F1 (Tm multiplexing). The 2nd strategy used induced fluorescence resonance energy transfer (iFRET) to detect DEN-1 in channel F2 and DEN-3 in channel F3 (color multiplexing). We incorporated fluorogenic primers specific to DEN-1 and DEN-3 at the ends of the amplicons by means of the PCR. SYBR Green I (SGI) molecules (Molecular Probes/Invitrogen) intercalating with the duplex amplicons acted as an energy donor for the acceptor fluorophores tagged to the primers. As a result, light with a longer wavelength was emitted from the excited acceptor fluorophore. During melting-curve analysis, the progressive rise in temperature diminished the interaction between SGI and acceptor fluorophore as the duplex amplicons dissociated. A dramatic reduction in the fluorescence signal occurred at the Tm of the amplicon.
primer design
We retrieved complete genome sequences of DVs (n = 112) from GenBank and aligned them with MAVID (17). The moderately conserved 3' untranslated region was selected for primer design (Fig. 1A
) with the OLIGO software (Molecular Biology Insights). All primers were purchased from Integrated DNA Technologies. The 4 serotypes were amplified with 4 serotype-specific forward primers and a common DV-specific reverse primer (Table 1
; Fig. 1B
). Forward primers for DEN-1 and DEN-3 were labeled at the 5' end with BODIPY 630/650 (Molecular Probes/Invitrogen) and Cy5.5, respectively. We used deoxyinosine in the DEN-2 forward primer (D2F) to overcome the genotypic variations within DEN-2. A GC tail was added to the 5' end of the DEN-4 forward primer (D4F) to give a product Tm that was higher than that of DEN-2 (predicted difference, 3.6 °C; Table 1
). Because 9 of 10 consecutive nucleotides upstream of the 3' end of the D4F primer were identical among all 4 DV serotypes (Fig. 1A
, asterisks), we applied a simple strategy of sequence-specific PCR for accurate typing of DEN-4 to minimize nonspecific annealing of primer D4F to non–DEN-4 templates. The specificity of primer D4F was enhanced with a deliberately destabilizing mismatched G base close to the 3' end of the primer (18). This change ensured that only DEN-4 was amplified under stringent PCR conditions. The amplicons were between 103 and 111 bp in length (Table 1
). A BLAST analysis demonstrated all primers to be specific for DV.
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Figure 1. Primer design for the 1-step RT-PCR LightCycler assay.
(A), alignment of amplified regions of DEN-1 to DEN-4 and positions of forward and reverse primers. Underlined sequences indicate the primer regions, and asterisks indicate the bases conserved among the 4 serotypes. (B), alignment of primer regions of all primers. Bo, BODIPY 630/650 fluorophore; I, deoxyinosine base; Cy, Cy5.5 fluorophore; Tail, GC tail (5'-CGC GCC GGC CGC GG-3'). There are 112 aligned DV genome sequences: 31 DEN-1, 52 DEN-2, 25 DEN-3, and 4 DEN-4. The numbers of viral sequences perfectly matching or possessing 1–2 mismatches with the respective forward primers (D1F to D4F) and the common reverse primer D1–4R, their proportions, and the percentages within each serotype are indicated to the right of the aligned sequences. In all forward primers, the 8 bases (AAGCTGTA) highly conserved in dengue viral genomes are underlined. Of the 112 aligned genome sequences, only 1 DEN-1 and 1 DEN-3 genome differ from this consensus sequence (by a single base). In the D4F primer, the boldface G base (near the 3' end of the primer and the 3' base of the 8-base consensus sequence) is a deliberate mismatch with the genome sequence to enhance the DEN-4 specificity of this primer. Most (96%) of the genome sequences show a perfect match with the common reverse primer, and only 4 genome sequences (4%) show a single mismatch.
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1-step rt-pcr assay
Using the Qiagen QIAamp viral RNA extraction reagent set, we extracted 24 RNA samples of known DV serotypes (n = 6 for each serotype) and 1 RNA sample of Japanese encephalitis virus from cell culture supernatants or clinical serum samples (Table 2
). We confirmed the presence of DV in the clinical samples with a separate nested RT-PCR assay (19). The RT-PCR assay in this study was performed with the LightCycler RNA Amplification Kit HybProbe (Roche Diagnostics) in glass capillaries in a LightCycler 1.5 System. The 20-µL reaction mixture contained the following components: 1x Roche LightCycler RT-PCR reaction mix HybProbe, 5 mmol/L MgCl2, 0.5 µmol/L D1–4R primer, 0.4 µmol/L D1F primer, 0.3 µmol/L each of D2F and D3F primers, 0.5 µmol/L D4F primer, 1x SGI, and 0.5 µL RNA sample. The 1x SGI concentration was used as suggested in the protocol of the LightCycler RNA Amplification Kit SYBR Green I. Reaction conditions were as follows: an RT step at 55 °C for 30 min and denaturation at 95 °C for 1 min, followed by 45 PCR cycles of 95 °C for 10 s, 59 °C for 15 s, and 72 °C for 20 s. The assay ended with a melting-curve analysis: heating to 95 °C without a hold, rapid ramping to 59 °C and holding for 30 s, gradual heating to 95 °C at 0.1 °C/s, and final cooling to 40 °C. The identities of the specific products were confirmed by agarose gel electrophoresis and DNA sequencing with an ABI 3130 Genetic Analyzer (Applied Biosystems). We determined the detection limit of each serotype by 10-fold serial dilutions of RNA samples extracted from cell culture supernatants with known viral titers. The viral titers for DEN-1, DEN-2, DEN-3, and DEN-4 were 3.27 x 106, 2.10 x 108, 1.63 x 107, and 1.16 x 108 plaque-forming units/L, respectively.
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Results
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The RT-PCR assay specifically detected the 4 DV serotypes, with no cross-reactivity between the primers (Fig. 2A
). The Japanese encephalitis virus was not amplified by the assay. Twelve (75%) of the 16 clinical serum samples known to be DV positive were accurately identified and serotyped. Channel F1 gave melting peaks due to SGI fluorescence for all 4 serotypes. In particular, the Tm difference between DEN-2 and DEN-4 was 4.7 °C, which was sufficient to differentiate these 2 serotypes. DEN-1 and DEN-3 had very similar Tms in channel F1; however, DEN-1 was characterized by its Tm (83.3 °C) in channel F2 owing to the iFRET between SGI and BODIPY 630/650, and DEN-3 was characterized by its Tm (83.9 °C) in channel F3 because of the iFRET between SGI and Cy5.5.
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Figure 2. Characteristics of the 1-step RT-PCR LightCycler assay.
(A), derivative melting curves showing the melting peaks characteristic of the 4 serotypes detected in 3 LightCycler channels (F1–F3). NTC, no-template control. Amplicon Tms are measured in channel F1 (SGI fluorescence) for DEN-2 and DEN-4 (i), in channel F2 (BODIPY 630/650 fluorescence) for DEN-1 (ii), and in channel F3 (Cy5.5 fluorescence) for DEN-3 (iii). In channel F3, inverted melting peaks due to DEN-2, and sometimes to DEN-1 and DEN-4, may be detected. The reason for these inverted peaks is not known; however, they do not interfere with DEN-3 typing. (B), calibration curves for the 4 serotypes. PFU, plaque-forming unit.
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The measurement of Tm was very precise, with within-run CVs ranging from 0.1% to 0.3% and between-run CVs ranging from 0.2% to 0.6% (Table 1
). The detection limits in plaque-forming units per reaction and the corresponding threshold cycle (Ct) value for DEN-1 (1.64 x 10–4 and 30.8) and DEN-3 (8.15 x 10–4 and 27.8) were 1 log lower than those for DEN-2 (1.05 x 10–3 and 33.7) and DEN-4 (5.80 x 10–3 and 30.7). The assay had a dynamic range of detection (103–108 plaque-forming units/L). The correlation coefficients for the correlation between viral concentration and Ct value were –0.99 for both DEN-1 and DEN-3, –0.98 for DEN-4, and –0.95 for DEN-2 (Fig. 2B
). Amplification plots are available (see figures in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue4). Amplification efficiency was highest for DEN-1 and lowest for DEN-4, as calculated from the following equation: efficiency = 10(–1/slope) (20).
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Discussion
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Our assay was based on Tm and color multiplexing for the detection and typing of DV. The unique amplicon Tm of each serotype in different channels (F1 to F3) allowed specific detection of DV. Adding a GC tail improves the detection of single-nucleotide polymorphisms (21)(22) and has a marked effect on Tm differentiation for small amplicons (21)(23), such as the 
100-bp amplicons in this study. By contrast, minor base changes within amplicons typically do not produce detectable Tm changes. Because the amplicon sequence is highly conserved within the same DV serotype, a 4.7 °C Tm difference would be able to accommodate the possible minor genotypic variations in DEN-2 and DEN-4.
As expected, the amplicons from DEN-1 and DEN-3 had very similar Tms (Table 1
) and thus could not be distinguished in channel F1. Classically, 2 hybridization probes (1 donor and 1 acceptor) are required for fluorescence resonance energy transfer to occur. Genotypic variations in the genome of the same serotype, however, make it difficult, if not impossible, to design 2 hybridization probes for specific detection and typing. This study combined fluorogenic primers and SGI to achieve the same goal via iFRET. In fact, SGI is an effective energy donor for fluorescence resonance energy transfer, and the fluorescence generated by iFRET is 40 times greater than with the classic fluorescence resonance energy transfer (24). This assay produced distinct melting peaks due to BODIPY 630/650 in channel F2 for DEN-1 and due to Cy 5.5 in channel F3 for DEN-3. We chose these 2 less expensive dyes instead of LC Red 640 and LC Red 705, respectively, to reduce costs (25). A 1-step LightCycler assay based on the biprobe system and developed to detect only DEN-2 (26) is similar in principle to the iFRET assay.
Four (25%) of the serum samples known to be DV positive were not detected by this assay, but these false-negative samples tested either negative or weakly positive in the 1st-round PCR of a nested RT-PCR assay, which is a 2-step nonquantitative assay. These results indicated that the DV titers in the samples might be too low to be detected. The RNA sample volume per reaction was believed and then confirmed to be the major factor affecting the sensitivity of our assay. The input sample volume for the nested RT-PCR assay was 10-fold larger than that for our assay. The sensitivity of our assay was improved by 12.5% (10 vs 12 of 16 samples) by increasing the RNA sample volume from 0.5 to 5 µL. This result showed that the sample-volume effect became magnified when the viral load in the serum was low. We suggest the use of larger sample volumes for clinical samples. For precious RNA samples from suspected DV-positive patients, we suggest a 0.5-µL input sample volume for screening, followed by confirmation of a negative result with a larger sample volume.
The present assay gave a PCR efficiency >2, which is beyond the maximum theoretical value (efficiency = 2; slope = –3.3). The PCR efficiency was calculated from the slope of the calibration curve, –2.78 to –2.27 in this assay, which was generated from the Ct values of a dilution series of DV RNA. The presence of primer dimers in the reaction mixture increased the overall SGI signal, which in turn lowered the Ct values and yielded a PCR efficiency >2. Similar findings were noted in a recent study that used a Roche Diagnostics reagent set for amplifying Chlamydia pneumoniae in the LightCycler (27). Nevertheless, the sensitivity and specificity of our assay were not affected, because the PCR efficiency compared favorably with those of other available RT-PCR assays for dengue diagnosis (7)(13).
Positive controls for all 4 serotypes and a no-template negative control should be run in parallel with test samples. This practice not only provides a reference for interpretation but also helps to monitor the accurateness of DV detection and typing, which is particularly important to recognize if deterioration of reagents has occurred. We once observed a systematic difference in results when we used a new batch of reagent sets; this systematic difference led to a Tm shift in all channels and for all serotypes. The Tm shift was determined to have been caused by improper storage conditions during delivery. When the samples were retested with another batch of the reagent sets that had been delivered under proper storage conditions, the Tm shift was negligible. Variations between different batches of reagent sets have previously been reported and could lead to a 2.5-fold difference in mRNA quantification (28).
The entire assay requires 2 h (40 min for preparation plus 80 min for RT-PCR reaction/melting-curve analysis), compared with 
3 h for the conventional TaqMan assays (11)(13). Our assay is also more economical, because the primers are used for both detection and typing, only 2 primers have to be labeled with fluorophores, and no additional fluorescence probes are required.
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Acknowledgments
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This study was supported by Internal Competitive Research Grant G-YD97 of The Hong Kong Polytechnic University. Purchase of the 3130 Genetic Analyzer was supported by funds from The Hong Kong Polytechnic University (1.55.27.DD02). The DV RNA samples were supplied by Public Health Laboratory Services Branch, Centre for Health Protection, Hong Kong SAR, China. Disclosures: None declared.
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Footnotes
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1 Nonstandard abbreviations: DV, dengue virus; RT, reverse transcription; Tm, melting temperature; iFRET, induced fluorescence resonance energy transfer; SGI, SYBR Green I; Ct, threshold cycle. 
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Abstract
Introduction
