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

Date Published: July 10, 2019

Publisher: Public Library of Science

Author(s): Jih-Jin Tsai, Wei-Liang Liu, Ping-Chang Lin, Bo-Yi Huang, Ching-Yi Tsai, Pei-Yu Alison Lee, Yun-Long Tsai, Pin-Hsing Chou, Simon Chung, Li-Teh Liu, Chun-Hong Chen, Yu Ru Kou.


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.

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.

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.

Partial Text

Dengue virus (DENV), a member of the genus Flavivirus in the family Flaviviridae, causes dengue in humans. Dengue virus infection, one of the most important and mosquito-borne viral diseases, is considered a major public health problem in developing tropical and sub-tropical countries mainly in Asia, South America, and the Caribbean with major disease outbreaks and endemic every year [1]. Four serotypes of dengue virus (DENV-1, -2, -3, and -4) have been identified, with distinct genotypes within each serotype. Multiple virus serotypes have been found co-circulating in the hyperendemic regions in Southeast Asia and Pacific [1, 2]. Its single-stranded, positive-sense genomic RNA contains 5′- and 3′- untranslated regions (UTR) and a single open reading frame encoding for a single polyprotein that could be cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).

Testing with the pan-DENV RT-iiPCR, the analytical and clinical performance of the fully automatic POCKIT Central system was comparable to those of the semi-automatic POCKI combo system, which was validated previously to offer performances equivalent to the CDC DENV1-4 real-time RT-PCR for the detection of DENV in human serum [7, 24, 26]. Validation with 60 serum samples prepared from dengue-suspected patients demonstrated that the pan-DENV RT-iiPCR performed equally well on both the semi-automated and fully automated equipment (100% agreement; CI95%, 98.81 ~ 100%; κ = 1.0; Table 3). Moreover, the pan-DENV RT-iiPCR can work on both systems to detect the four DENV serotypes in DENV-infected mosquitos (Table 4).




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