Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported to WHO as an outbreak in Wuhan City, Hubei Province, China on end of 2019, afterwards epidemic in many countries, and pandemic on the worldwide in 2020. Usually detection of coronavirus including SARS-CoV-2 was detected by real-time RT-PCR method, but it must be long time that RNA is treated by extraction, concentration and purification, and detected by RT-PCR method. We modified various methods, of which evaluated if each method is short and simple enough. In one point of the evaluations, real-time RT-PCR could be finished in very short time with using mobile real-time PCR device PCR1100 (Nippon Sheet Glass Co. Ltd.). It was able to detect positive control RNA for 20 minutes by each method according to the National Institute of Infections Disease in Japan (NIID), and less than 13.5 minutes according to the Centers for Disease Control and Prevention in USA (CDC). In another point of the evaluations, surprisingly, Human coronavirus 229E, which was substituted for SARS-CoV-2, could be detected in crude state without treatment in advance of RNA. As that was, it was possible to detect coronavirus with direct RT-PCR. Therefore, it might eliminate wasteful time, avoid secondary infection and risk of contamination. In light of the above two points, SARS-CoV-2 might be detected more quickly and more simply. With using this mobile real-time PCR, these methods should be suitable for not only SARS-CoV-2 but also other various viruses and might save time compared to earlier detection methods.

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.