Several real-time PCR based methods for detection of DENV have been reported in the last decade. These assays have targeted the 3'UTR [
9,
15], NS5 [
11,
16], core [
9] and the envelope [
17] gene sequences. Majority of the recent reports describe development of the serotype specific real-time PCR for dengue using TaqMan probes [
8,
18] or FRET probes [
6]. Though these methods are useful for serotyping of DENV they may not be cost-effective for routine diagnosis as only a small percentage of samples are positive for DENV RNA during the non endemic season and during the active transmission season only about 50% of samples may be positive for DENV RNA [
6]. A group specific PCR would be a useful tool for initial screening of the samples and only those samples positive for DENV can then be subjected to serotyping. Lai et al., [
6] initially screened samples by a SYBR green based group specific real-time PCR and then serotyped the positive samples by a duplex or a fourplex TaqMan based assay, thereby reducing the operation cost on diagnosis by half. The SYBR green-based detection systems have the major disadvantage of false positives due to dye binding to primer dimers or to DNA amplified non-specifically. Melt curve analyses is often used to confirm the fidelity of the reaction. Other group specific real time PCR assays reported are also SYBR green based [
9,
10,
19]. The first DENV group specific assay using TaqMan probes was described by Callahan et al., [
11]. In the assay the authors have used multiple probe primer sets for establishing a group specific RT-PCR assay. This could have been because of lack of a suitable stretch of conserved nucleotides for designing the conventional TaqMan probes, earlier observed by Laue et al., [
16]. The new generation MGB probes can be much shorter in length because of the Minor groove binder tagged to the probe, which increases the thermal stability of the probes. We used the TaqMan MGB probe to develop the group specific assay in this report which made it possible to use the short conserved region in the 3'UTR of the dengue genome to design the assay using a single probe and primer pair combination. The fluorescent probes in TaqMan assay are known to be target specific and sensitive to mismatches [
20]. To avoid false negative results due to mismatches in the probe binding sites, the 3'UTR region of thirteen Indian strains, representing all four serotypes, isolated from different regions in India were sequenced. These sequences were aligned with representative sequences of all four serotypes from the GenBank and a conserved region of 100 nucleotides was used to design the primers and probe.
All four serotypes of DENV from infected mouse brain or cell cultures or mosquitoes or clinical samples could be detected by the qRT-PCR. No amplification with the related Flaviviruses or with samples from other febrile illness was observed. The sensitivity of the qRT-PCR in terms of PFU was highest for DENV-2 at 0.001 PFU followed by DENV-1 and DENV-3 at 0.01 PFU and DENV-4 had the lowest sensitivity of 1 PFU. The difference in sensitivity could have been due to a difference in the proportion of non-infectious RNA transcripts to infectious particles. The mismatches between the DENV-4 virus sequence and the reverse primer could have contributed to the lowest sensitivity of 1 PFU. An alternate reverse primer with DENV-4 sequence can be included to bring the sensitivities at par. The qRT-PCR assay was able to detect viral RNA in a significantly larger number of clinical samples (30.7%) than RT-PCR (16.9%) (p < 0.001, Mc Nemar's test). The qRT-PCR also detected DENV RNA in a larger number of IgM positive samples when compared to the nested RT-PCR. There is increasing recognition of the importance of viral burden in the pathogenesis of DHF and a direct association between viral load and severity of the disease has been reported [
21‐
23] In earlier studies on viral load the method used to quantitate the virus in the blood samples were either mosquito infectious dose (MID
50) or PFU. It has been difficult to compare any two studies as the measures vary depending upon the strain of the virus and the substrate used i.e. mosquito or cell line. None of the group specific real-time PCRs reported [
6,
9‐
11,
24] developed a quantitative RNA standard. We developed an RNA standard for quantitating DENV RNA in this study. The RNA copy number, which offers minimum variability, was used to quantitate the viral load in human clinical samples. Viremia was found to be higher in the initial days of the illness decreasing gradually during the later stages of the infection. This result is consistent with earlier findings based on mosquito inoculation and serotype specific real-time PCR [
15,
25].
An important aspect of dengue disease control is vector surveillance. Detection of DENV positive mosquitoes will be useful to monitor the infection rates within vector mosquito population and provide an early warning signal to predict an impending epidemic [
26]. Currently IFA, insect bioassay, ELISA and RT-PCR are the methods available for detection of DENV in mosquitoes [
27]. The qRT-PCR assay could detect DENV-2 RNA in all the inoculated mosquitoes. The sensitivity of detection was such that a 40
th fraction of the infected mosquito body lysate was sufficient to give a positive signal in the qRT-PCR, thus even low levels of viral RNA in infected mosquitoes should be detected with the assay. No false positives were observed with control mosquitoes. However testing of field caught mosquitoes by the qRT-PCR will prove its usefulness for vector surveillance.