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.

Evaluation of rapid diagnostic tests to detect dengue virus infections in Taiwan

“…RT-PCR has been recommended for confirmational diagnosis of dengue virus infection, allowing for early intervention for surveillance, outbreak investigations, and clinical management. In our previous studies, reverse transcription insulated isothermal PCR (RT-iiPCR) using pan-DENV or singleplex DENV-1–4 serotyping reagents in a mobile semi-automated taco mini/Pockit system or a fully automated sample-to-answer POCKIT Central system were validated for rapid detection of dengue virus in human serum in approximately 90 minutes, reaching analytical sensitivity of 1 to 10 PFU/ml of serum for the four serotypes of dengue virus [ 53 – 55 ]. However, the disadvantages of the nucleic acid test are that serum samples must be shipped to a central laboratory, and the experiments must be performed by well-trained medical technician to detect DENV.…”

Dengue Detection: Advances in Diagnostic Tools from Conventional Technology to Point of Care

“…However, the conventional PCR requires skilled personnel and lab facilities to perform this manual assay. To automatize the sample extraction to amplification, an insulated isothermal PCR assay has been developed to detect DENV [95]. They developed POCKIT combo central system which accommodates a cartridge where serum samples are loaded and the cartridge contains all the extraction, amplification reagent and the POCKIT system automatically provides the qualitative result with a lowest detection points of 1 and 10 PFU/mL for DENV-1,3 and DENV-2,4 respectively.…”

Dengue Virus and Yellow Fever Virus Detection Using Reverse Transcription–Insulated Isothermal PCR and Comparison with Real-Time RT-PCR

“…However, additional instruments are available for automated RNA extraction and sample-to-answer detection based on this technology. 11,12 In conclusion, the POCKIT DENV and YFV reagent sets compared favorably with sensitive rRT-PCRs for each pathogen, and these may provide accurate and lesscomplex options for molecular testing in certain laboratory settings.…”

“…3,4,6,8 Recently, a hydrolysis probe-based reverse transcriptioninsulated isothermal PCR has been developed for the detection of DENV in serum (POCKIT DENV Reagent Set, GeneReach Biotechnology, Taichung City, Taiwan). [9][10][11][12][13] This assay can be performed on a handheld battery-powered instrument that is inexpensive relative to the costs of real-time PCR instruments. Although assay comparisons between the DENV reagent set and an FDA-cleared DENV rRT-PCR have been reported, 9,10 independent evaluations of this assay have not been published.…”