Direct detection of Dengue viruses without extraction of RNA on the mobile real-time PCR device

Abstract

Dengue virus (DENV) is the cause of dengue / severe dengue and a virus of the Flaviviridae family, furthermore, dengue fever has rapidly spread in the world in recent decades. DENV is transmitted by female mosquitoes, mainly of the specie Aedes aegypti. The main method to control or prevent the transmission of DENV is to combat the mosquito vectors. Among these, one of important methods is to monitor the DENVs in the mosquito vectors. For the detection of DENV, nucleic acid amplification tests (NAAT) were recommended, of which criterion standard is real-time RT-PCR with highly sensitive and specific. However, it takes long time as to judge the result per a reaction, besides the necessity of the treatment of RNA in advance, example of extraction, concentration and purification. It was our object in this time to develop the method of real-time RT-PCR detecting DENVs in shorter time, moreover without especial treatment of RNA from the mosquito in advance. Besides, this work was performed with combing the mobile real-time PCR device with the one-step RT-PCR reagent. Firstly, we succeeded in shortening the time of real-time RT-PCR for the detection of DENV per one reaction, so that the judgement needed less than 20 minutes if genomic RNA treated in advance. Moreover, each value on the real-PCR device was quantitatively correlated with the positive control RNA from 1.0 x 10 ^ 3 copies to 1.0 x 10 ^ 0 copies per reaction (This correlation coefficient R2 > 0.95). Additionally, it made sure that this method could be applied to each DENV serotype. Secondly, we established the basis of procedure for the real-time RT-PCR without the treatment in advance so-called "direct". As the result that the positive control RNA additive was utilized instead of the real DENV, spiked into the mosquito homogenized and sampled the supernatant without treatment, it was possible to detect on the real-time RT-PCR even if mosquitoes immediately after blood-feeding. For this reason, this method might be able to utilize in human sera, too. According to the results of this work, we could suggest the method is possible to detect DENV more quickly and more simply than heretofore. The Real-time "direct" RT-PCR, especially, could be performed with mobile real-time PCR PCR1100 device and one step RT-PCR reagent only. This method must help to detect some viruses other than DENV, too.

SERS-Based Colloidal Aptasensors for Quantitative Determination of Influenza Virus

“…However, PCR requires several hours to perform 30–40 cycles. Several attempts have been made to create rapid PCR within small portable devices; their robustness and accuracy are to be studied in the near future [ 4 ]. Antibody-based assays have been used for rapid diagnostics of influenza A, HIV and other viruses.…”

Point of care testing for detection of coronaviruses including SARS-CoV-2 from saliva without treating RNA in advance

“…It was our purpose this time to contribute to develop POCT for microbes such as SARS-CoV-2, and thus needed to improve NAATs method. Therefore, it was first focused on the mobile rRT-PCR device ’PCR1100’ s and rRT-PCR reagent kit and evaluated whether they were ’rapid and easy’, similarly to recently other reports for detection of SARS-CoV-2 [12] and dengue virus [22]. Next, it was considered whether rRT-PCR could be performed in saliva specimen or not without treating RNA extraction, etc.…”