Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid

Abstract

Polymerase chain reaction (PCR) is an important molecular biology technique for in vitro amplification of nucleic acids. Reverse transcriptase quantitative PCR (RT-qPCR) and more recently reverse transcriptase digital droplet PCR (RT-ddPCR) have been developed for the quantification of nucleic acids. We developed an RT-ddPCR assay for the quantification of attenuated dengue virus serotype 2 nucleic acid and compared it with a routine RT-qPCR assay. While the routine RT-qPCR assay targets the NS5 gene, the E gene was selected for the optimization of the RT-ddPCR assay conditions. The specificity of the assay was demonstrated using the attenuated dengue virus serotype 2 alone and in the presence of the other three dengue serotypes. The results from both assays for 25 samples of the attenuated dengue virus serotype 2 were found to be comparable, with an R from the linear regression analysis of >0.98. A major advantage of the RT-ddPCR assay is that it allows quantification of nucleic acid, without the need of a standard curve. RT-ddPCR can be implemented for the absolute quantification of dengue vaccine virus nucleic acid during the vaccine manufacturing process.

Current challenges in biotherapeutic particles manufacturing

“…For genome particles quantification, real time polymerase chain reaction (qPCR) is usually the method of choice. Droplet digital polymerase chain reaction (ddPCR) has also been recently used for quantifying viral genome, without the need for a standard curve [154,155]. Depending on the final use of virus-based particles, and different from mAbs, infectivity can be a critical parameter.…”

Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool

“…Virus spike sample quantification using RT-ddPCR. The viral load of the 15 spiked mosquito pool samples was determined using reverse transcription droplet digital PCR (RT-ddPCR), a highly sensitive method that allows absolute quantification without the need for a standard curve 42 . The primer and probe sequences used can be found in Table S2.…”

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression

“…Also, it has been proved that dd-PCR is more sensitive to detect rare targets at low copy numbers than the RT-qPCR. 26,27 In cell experiments, detection sensitivities of dd-PCR and RT-qPCR were identified, with the conversion between results of the two methods based on the same amount of template results as Y = 33. In the present study, within 3 months before loss of MR4.0, 4 of the 10 patients who loss of MR4.0 presented markedly increased dd-PCR results compared with those of RT-qPCR.…”