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A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 107 PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.
Dengue virus is a mosquito-borne flavivirus and the most prevalent arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America (7). Dengue virus belongs to the family Flaviviridae. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness to severe and fatal hemorrhagic disease (2, 18). There are four distinct serotypes—designated DEN-1, DEN-2, DEN-3, and DEN-4—which can be distinguished by serological and molecular methods. The flavivirus genome is approximately 11 kb in length, and the complete genome sequence is known for isolates of all four dengue virus serotypes. The genome is composed of three structural protein genes, encoding the nucleocapsid or core protein (C), a membrane-associated protein (M), an envelope protein (E), and seven nonstructural (NS) protein genes (4). The order of proteins encoded is 5′-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′.
The two basic methods used by most laboratories for the diagnosis of dengue virus infection are detection of virus or antidengue antibodies. For convalescent-phase sera, serological diagnosis of antibodies based on capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) has become the new standard for the detection and differentiation of primary and secondary dengue virus infection (21). However, antibody detection has less impact on patient management and control measures exercised by medical and public health personnel. Therefore, there is a great demand for the rapid detection of dengue virus infection in the acute phase of illness in order to provide timely clinical treatment and etiologic investigation and disease control.
For virus detection, virus isolation by cell culture remains the “gold standard” although it has the disadvantage in that longer than 7 days is usually required to complete the test. Recently, two different methods had been demonstrated to be useful in the detection of dengue virus. These include genomic sequence detection by RT-PCR and dengue virus antigen detection by ELISA (1, 21). Several laboratories have published various RT-PCR protocols for dengue virus identification (8, 9, 10, 14, 17, 19, 20). Among these, the two-step nested RT-PCR protocols originally reported by Lanciotti et al. and later modified to single-step multiplex RT-PCR for the detection and typing of dengue virus by Harris et al. are well known (9, 14). These assays used dengue core-to-premembrane gene regions as its target sequence for dengue virus detection. It had the advantage of detecting and differentiating four dengue serotypes by analyzing the unique sizes of amplicons in the agarose gel. More recently, several investigators have reported fully automatic real-time PCR assays for the detection of dengue virus in acute-phase serum samples (3, 5, 11, 15). The real-time PCR assay has many advantages over conventional RT-PCR methods, including rapidity, quantitative measurement, lower contamination rate, higher sensitivity, higher specificity, and easy standardization. Thus, nucleic acid-based assays or real-time quantitative assay might eventually replace virus isolation and conventional RT-PCR as the new gold standard for the rapid diagnosis of virus infection in the acute-phase serum samples.
In an attempt to develop a simple, reliable, and universal RT-PCR protocol to systemically detect and differentiate various arbovirus, we have recently developed a real-time quantitative RT-PCR system based on SYBR Green I DNA dye-binding fluorophore. The rationale is to develop a diagnostic system with an automated platform using real-time PCR equipment and a universal RT-PCR protocol that allows multiple primer sets designed and tested without the need to change the RT-PCR conditions. Theoretically, this approach will increase the successful rate in generating a panel of optimal primer pairs. In addition, all the primer pairs can be run simultaneously in a single unit of a real-time RT-PCR instrument. Indeed, G. Kuno had originally proposed this concept using conventional RT-PCR in the detection of arbovirus (13). In the present study, we report the development of a group- and serotype-specific one-step SYBR Green I-based real-time RT-PCR assay by Mx4000 for the screening and typing of dengue virus RNA. The results demonstrated that the one-step SYBR Green I-based real-time RT-PCR assay is more sensitive than the standard cell culture method.
The following dengue virus nucleotide sequences were retrieved from GenBank (accession numbers shown in parentheses): DEN-1 West Pac (U88535), DEN-1 16007 (AF180817), DEN-1 S275/90 (M87512), DEN-2 New Guinea C (AF038403), DEN-2 16681 (U87411), DEN-2 JAM (M20558), DEN-2 FJ-10 (AF276619), DEN-3 H87 (M93130), DEN-3 MALAY94-3 (AB010990), DEN-3 80-2 (AF317645), DEN-3 CH53489 (AF008555), DEN-4 H241 (M14931), and DEN-4 China Guangzhou B5 strain (AF289029). Nucleotide sequences were aligned to identify conserved regions using DNA Star software (Perkin-Elmer, Norwalk, Conn.). Potential target regions were selected and primers were synthesized and evaluated for quantitative RT-PCR. The final sequences of the group- and serotype-specific primer pairs are shown in Table Table11.
Viral RNAs were prepared for four dengue virus serotypes, including 4 prototype strains (DEN-1, Hawaii; DEN-2, New Guinea C; DEN-3, H87 [Philippines]; and DEN-4, H241 [Philippines]) and 12 local isolates from imported or indigenous dengue patients during 1987 to 2000 as listed in Table Table2.2. Viruses were propagated in Vero cells and viral titers were determined by plaque forming assay (6). Viral RNAs (70 μl) were extracted from 140 μl of cell culture supernatants or serum samples using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and stored at −70°C.
One-step SYBR Green I-based RT-PCR amplification was performed in the Mx4000 quantitative PCR system (Stratagene). After optimization of each of the primer pairs, samples were assayed in a 50-μl reaction mixture containing 10 μl of sample RNA and optimal concentration of each of the primers by using the Brilliant single-step quantitative RT-PCR core reagent kit (Stratagene) with MgCl2 at a 3 mM final concentration, dimethyl sulfoxide at a 3% final concentration, and 0.5 μl of SYBR Green stock solution (Stratagene SYBR Green QPCR core reagent kit) diluted 1:333 in dimethyl sulfoxide. The thermal profile for one-step SYBR Green-based RT-PCRs consisted of a 30-min RT step at 45°C and 10 min of Taq polymerase activation at 95°C, followed by 45 cycles of PCR at 95°C for 20 s (denaturation), 55°C for 30 s (annealing), and 72°C for 30 s (extension).
Following amplification, a melting curve analysis was performed to verify the correct product by its specific melting temperature (Tm). Melting curve analysis consisted of a denaturation step at 95°C for 1 min, lowered to 55°C for 30 s, and followed by 40 cycles of incubation in which the temperature is increased to 95°C at a rate of 1°C/30 s/cycle with continuous reading of fluorescence. Results were analyzed with the melting curve analysis software of the Mx4000. For SYBR Green-based RT-PCR amplification, amplification plots and Tm values were routinely analyzed to verify the specificities of the amplicons.
The serum samples used in this study were collected from patients with confirmed cases of dengue reported to the Department of Health, Centers for Disease Control and Prevention, during the period between 1998 and 2002. A confirmed case of dengue virus infection was defined as febrile illnesses associated with the isolation of dengue virus, positive RT-PCR result, or ≥4-fold increase of dengue-specific IgM or IgG antibody in paired serum samples. It should be emphasized that all of the 193 acute-phase serum samples analyzed in this study were selected from patients with cases of dengue confirmed by either positive dengue virus isolation or positive serological test (≥4-fold increase of dengue-specific IgM or IgG antibody in paired serum samples) without taking into account RT-PCR results. Sera collected during the period between days 1 and 7 after the onset of symptoms are referred to as acute-phase samples.
The isolation of dengue virus was performed using the mosquito cell line Aedes albopictus, clone C6/36. For each acute-phase serum, 4 μl of serum sample was diluted in 200 μl of cultured medium (RPMI, Gibco/BRL, Life Technologies) (containing 1% FCS) and added to a 96-well microtiter plate, 50 μl/well in quadruplicate. Then, 105 cells/100 μl/well of C6/36 were added into the microtiter plate and incubated at 37°C for 7 days. Cells were harvested and viruses were identified with dengue virus group-specific, serotype-specific monoclonal antibodies and fluorescein isothiocyanate-conjugated goat anti-mouse antibody.
A modified capture IgM and IgG ELISA was developed to measured the IgM and IgG antibodies of patients infected with dengue virus as previously described by Innis et al. (12). Each microtiter well was coated with affinity-purified goat anti-human IgM (μ-specific) or IgG (γ-specific) antibodies (5 μg/ml, 100 μl/well), followed by incubation with 100 μl of 1:100 diluted serum. Subsequent incubations were performed with 100 μl of cocktail containing 1:3 diluted pooled virus antigens from culture supernatants of DEN-1-, DEN-2-, DEN-3-, or DEN-4-infected Vero cells and MAb D56.3 (1 μg/ml), followed by incubation with 1:1,000 diluted alkaline phosphatase-conjugated goat anti-mouse IgG (γ-specific). Then, substrate p-nitrophenyl-phosphate was added, and the optical density taken at the dual wavelengths of 405 and 630 nm. A positive sample was defined as having a test absorbance/negative control ratio of ≥2.0, and a negative sample was defined as having a ratio of <2.0.
One of the advantages of SYBR Green-based real-time RT-PCR is that it is relatively easy to design and test primer pairs suitable for RT-PCR analysis. Primers were selected based on highly conserved regions of dengue virus genome. After nucleotide sequence alignment using DNA Star software (Perkin-Elmer), potential target regions were identified in the regions of core, NS3, NS5, and 3′ noncoding genes. More than 30 primer pairs were synthesized and screened for initial evaluation. Among these primers tested, a set of group- and serotype-specific primer pairs in the core gene region was found to be most sensitive. The primers were designed to take into account of mismatches among strains and to avoid primer-dimer formations. The final sequences and the genomic locations of these primers are shown in Table Table1.1. The condition of thermal profile for one-step SYBR Green-based RT-PCRs and the optimal concentrations of primer pairs were then optimized to increase the sensitivity and specificity. The optimal concentrations for each of the primer pairs were titrated and found to be 50 and 50 nM for the DN-F and DN-R pair (group specific), 50 and 50 nM for the DN-F and D1-R pair (DEN-1 specific), 50 and 50 nM for the DN-F and D2-R pair (DEN-2 specific), 50 and 25 nM for the DN-F and D3-R pair (DEN-3 specific), and 50 and 50 nM for the DN-F and D4-R pair (DEN-4 specific), respectively. The Tm values of primer-dimers were found to be below 75°C, whereas the Tm values of each of these amplicons from the group- and serotype-specific primer pairs were in the range of 79 to 83°C depending on the various strains tested. No primer-dimers were detected as demonstrated by melting curve analysis if optimal primer concentrations were followed.
To ascertain the detection limits of the one-step SYBR Green-based RT-PCR method using group- and serotype-specific primer pairs in dengue diagnosis, we tested 10-fold serial dilutions of seed viruses that had been quantitated by plaque forming assay. Figure Figure11 and Fig. Fig.22 showed the standard curves of four dengue prototype viruses using group- and serotype-specific primer pairs, respectively. The detection limit of each group-specific primer pair was between 4.1 and 43.5 PFU/ml for various dengue serotypes. The detection limits of the serotype-specific primer pairs were calculated to be 10 PFU/ml for DEN-1, 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4.
To verify that the dengue group- and serotype-specific primer pairs can be used to detect and differentiate the majority strains of four dengue virus serotypes, we had selected and tested 16 dengue virus strains from our virus bank. These included 4 prototype strains collected from the American Type Culture Collection and 12 local isolates from patients with imported or indigenous dengue during 1987 to 2000. Most of the strains had been sequenced at the core and envelope gene regions and could be assigned to distinct genotypes. Among these, D4 8700544 was an unique isolate which could neither be grouped into the DEN-4 genotype I nor II based on the sequences in the core and envelope regions (data not shown). Table Table22 showed the results of SYBR Green I-based one-step RT-PCR analysis. It was demonstrated that these group- and serotype-specific primer pairs were highly specific in the detection and differentiation of various dengue virus strains. None of these primer sets, however, amplified West Nile virus (Eg101 strain), yellow fever virus (17D vaccine strain), and Japanese encephalitis virus (JaGAr strain). This is in contrast to a panel of primer pairs with broad specificity to flavivirus or restricted specificity to West Nile virus, yellow fever virus or Japanese encephalitis virus, which had been shown to successfully detect and differentiate each of these corresponding viruses (data not shown). Figure Figure33 shows the results of agarose gels demonstrating that this group- and serotype-specific primer pairs are highly specific in the detection and differentiation of various strains since the amplicons generated have the correct size with expected sequences (data not shown). The 171 bp that appear in Fig. Fig.33 were the expected products for all four dengue serotypes using group-specific primer pair (DN-F/DN-R), whereas the 193-, 204-, 204-, and 132-bp amplicons were the expected products for each of the DEN-1, DEN-2, DEN-3, and DEN-4 serotype, respectively, using type-specific primer pairs. The 375-bp amplicon appeared in Fig. Fig.3A3A was found to be a longer product sharing the same sequences with 171-bp amplicon at its 5′ end with additional DEN-1 sequences following it.
To evaluate the SYBR Green I-based one-step RT-PCR in the clinical diagnosis of dengue virus RNA, we analyzed 193 acute-phase serum samples collected from confirmed dengue patients during the period between 1998 and 2002. These acute-phase serum samples selected for analysis were collected from confirmed dengue patients with either positive dengue virus isolation or positive serological test without taking into account of RT-PCR result. The results are shown in Table Table33 and revealed that 83 versus 67% out of 193 acute-phase serum samples tested were positive for one-step SYBR Green-based RT-PCR method and cell culture method, respectively. The positive rates for each of four dengue serotypes were found to be 63% for DEN-1, 67% for DEN-2, 77% for DEN-3, and 60% for DEN-4 using the virus isolation method, versus 75% for DEN-1, 82% for DEN-2, 92% for DEN-3, and 100% for DEN-4 using the one-step SYBR Green-based RT-PCR. Further analysis showed that the sera that were dengue virus negative by virus isolation and dengue virus positive by the SYBR Green-based RT-PCR assay had relatively fewer amplicons than those samples that were positive by both virus isolation positive and the SYBR Green-based RT-PCR assay (Fig. (Fig.44).
To further address the detection limit of the one-step SYBR Green-based RT-PCR, we had reclassified the above 193 serum samples with the criterion of presence or absence of dengue-specific IgM and/or IgG antibodies. The results are listed in Table Table4.4. Compared with the cell culture method, the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific IgM and/or IgG antibodies as the cell culture method. Two factors may account for the lack of sensitivity of the cell culture method: (i) a greater volume of serum samples is available (4 μl for cell culture versus 20 μl for real-time RT-PCR was added for each reaction, respectively) (Fig. (Fig.4)4) and (ii) real-time RT-PCR is less influenced by neutralizing IgM and/or IgG antibodies than cell culture method (Table (Table4).4). This result is very encouraging and suggests that one-step SYBR Green-based RT-PCR could replace the conventional cell culture method as the new gold standard in future clinical diagnosis of dengue virus infection.
The application of PCR in molecular diagnosis has gradually replaced traditional cell culture method as the gold standard for virus detection. The real-time PCR assays have many advantages comparing to conventional PCR including simplicity, rapidity, sensitivity, and low contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR (16). Among these, the most widely used format is the 5′→ 3′ nuclease oligoprobe (TaqMan assay) although this is most likely due to its commercial maturity. The TaqMan real-time PCR is highly specific due to the sequence-specific hybridization of the probe. Theoretically, it has the potential to develop multiplex up to four fluorophores in a single tube. This has led to a great expectation to analyze multiple pathogens and serotypes in clinical diagnosis. Ideally, a four-color multiplex protocol could be developed to detect and differentiate the four dengue serotypes using TaqMan one-step real-time RT-PCR. This goal, however, had not been realized at the present time.
Although the SYBR Green-based real-time PCR assays is less specific than the TaqMan real-time RT-PCR, it has the advantages of simplicity in primer design and universal RT-PCR protocols suitable for multiple target sequences. We have found that the optimization of primer concentration is critical in preventing primer-dimers and nonspecific amplification of other unrelated gene products. Indeed, nonspecific products such as primer-dimers and nonspecific amplification of other unrelated gene products could be occasionally detected at higher primer concentrations. However, these nonspecific products could be prevented if optimal concentrations were followed using the real-time RT-PCR protocol reported in this study. To assure the specificity of amplicons produced from SYBR Green I-based real-time RT-PCR in daily routine screening, both flavivirus-specific (FL-F, GCC ATA TGG TAC ATG TGG; FL-R, TGT CCC ATC CTG CGG TAT CAT [200 to 100 nM]) and dengue-specific primer pairs were used for each of the serum samples tested. Those serum samples positive for initial screening will then be tested for serotype by each of the four serotype-specific primer pairs. Excellent correlation was found among these six primer pairs over a thousand of serum samples tested so far. To further confirm the positive results of the SYBR Green-based RT-PCR method in clinical diagnosis, various confirmatory tests could be used. These include (i) virus isolation, (ii) TaqMan RT-PCR assay, and (iii) capture IgM and IgG ELISA to detect increased IgM and/or IgG antibodies using paired serum samples. We considered capture IgM and IgG ELISA the most suitable method for a confirmatory testing for the use of the SYBR Green-based RT-PCR method in clinical diagnosis due to its simplicity and reliability. In addition, most dengue virus laboratories routinely perform this test for their suspected dengue patients.
In an attempt to develop a universal diagnostic real-time RT-PCT protocol for arbovirus, we have taken an alternative approach using a SYBR Green I-based detection system. Kuno (13) was the first to propose a two-stage procedure for the systemic diagnosis of arbovirus using old conventional RT-PCR. In the first stage, broadly group-reactive primers are used to narrow the range of possible etiologic agents; in the second stage, virus-specific primers are employed for identification. It was emphasized that multiple group-reactive primers is essential to ensure the successful screening of a clinical sample against a multiple number of potential etiologic agents. Through careful design and testing, we have successfully developed a one-step RT-PCR system that can be used to detect and differentiate several flaviviruses, including dengue virus, Japanese encephalitis virus, Yellow fever virus, and West Nile virus (data not shown).
In the present study, we reported the development of group- and serotype-specific one-step SYBR Green I-based real-time RT-PCR assay for the screening and typing of dengue virus RNA. Analysis of clinical acute-phase serum samples demonstrated that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific IgM and/or IgG antibodies than cell culture method. The greatest advantage of this system is that one can actually detect and differentiate multiple viruses in a single run using the universal RT-PCR protocol with a panel of group- and type-specific primer sets. Our results suggest that one-step SYBR Green I-based RT-PCR system using the universal RT-PCR protocol should have great potential in the clinical diagnosis and epidemiological surveillance of viral diseases.
We thank Yun-Yih Chang, Hsiu-Ling Pan, and Chih-Heng Chen for their expert technical assistance.
This work was in part supported by grant NSC 89-2318-B-043B-001-M51 from the National Science Council and grant DOH90-DC-2015 from the Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China.