A fully automated sample-to-answer PCR system for easy and sensitive detection of dengue virus in human serum and mosquitos

Abstract

Background: The insulated isothermal PCR (iiPCR) technology enables consistent PCR amplification and detection in a simple heating device. A pan-dengue virus (DENV) RT-iiPCR, targeting the 5' untranslated region, was validated previously on the semi-automated POCKIT combo system (involving separate devices for nucleic acid extraction and PCR amplification/detection) to offer performance comparable to a laboratory real-time PCR. Working on the same technologies, a compact automated sample-in-answer-out system (POCKIT Central Nucleic Acid Analyser) has been available commercially for iiPCR, minimizing human error risks and allowing easy molecular bio-detection near points of need. Here, we evaluated the analytical and clinical performance of the pan-DENV RT-iiPCR on the fully automated system by comparison to those on the semi-automated system.

Methodology/principal findings: Testing sera containing serial diluted DENV-1, -2, -3, or -4 cell culture stock, the pan-DENV RT-iiPCR system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 PFU/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 PFU/ml, respectively, on the semi-automated system. Furthermore, both fully automated and semi-automated PCR system can detect all four DENV serotypes in mosquitos. Clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. Both systems detected the same 40 samples (ten DENV-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems.

Conclusions/significance: With performance comparable to a previously validated system, the fully-automated PCR system allows applications of the pan-DENV reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease.

Fig 1. Workflow of the semi-automatic and full-automatic PCR systems.

In the semi-automatic system, users add sample (serum, homogenized mosquitos) into the preloaded extraction plate (A) and place the plate into the nucleic acid extraction device for automatic nucleic acid extraction (B). The resulted nucleic acid extracts are added manually to the lyophilized reagent reconstituted first with provided buffer; the final PCR mixture is transferred manually to the reaction vessel (C). The reaction vessels are placed into the PCD device, which performs PCR amplification, detection and data interpretation automatically to provide qualitative results on the monitor (D). In the fully automatic system, users add serum sample to the preloaded extraction cartridge, place the extraction and reagent/consumable cartridges into the device, key in reagent and sample information, and start the assay (E). The device automates nucleic acid extraction, PCR reagent preparation, and PCR amplification, detection, and data interpretation to provide qualitative results on the monitor (F).