Application of One Step RT-PCR Assay for Detection of Flavivirus RNA in Mosquitoes

Hayashi, Akira; Kamakura, Kazumasa; Taga, Kenichiro; Mori, Hideto; Imura, Shunro; Eshita, Yuki; Uchida, Yukinori

doi:10.11150/kansenshogakuzasshi1970.77.822

Cited by 4 publications

(2 citation statements)

References 8 publications

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“…Furthermore, any method reported at the above need the treatment of RNA in advance such as concentration, purification and extraction, which must take more time and labour. Previously reported it possible to detect the DENVs in the mosquito by RT-PCR without treatment of RNA in advance, so-called direct RT-PCR [14,15]. After all, it would take more time and labour to judge results by means of not detecting by real-time.…”

Section: Introductionmentioning

confidence: 99%

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

Muraoka

Tanoi

Tada

et al. 2020

Preprint

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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.

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Section: Introductionmentioning

confidence: 99%

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

Muraoka

Tanoi

Tada

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Conventional virological methods for detecting flavivirus include virus isolation (Drury et al., 2001; Hoshino et al., 2009), immunofluorescent antibody assay (IFA) (Nagarkatti and Nagarkatti, 1980; Romero‐Vivas et al., 1998; Niedrig et al., 2008), reverse transcriptase polymerase chain reaction (RT‐PCR) (Kuno, 1998; Hayashi et al., 2003; Pyke et al., 2004) and real‐time RT‐PCR (RRT‐PCR) (Santhosh et al., 2007; Yang et al., 2010). Although RT‐PCR and RRT‐PCR methods are widely applied, reverse‐transcription loop‐mediated isothermal amplification (RT‐LAMP) is simpler, using only a water bath or heat block, and is highly efficient because the reaction is isothermal and requires no time for thermal changes (Notomi et al., 2000).…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Tembusu Virus by Reverse-Transcription, Loop-mediated Isothermal Amplification (RT-LAMP)

Tang¹,

Diao²,

Chen³

et al. 2011

Transboundary and Emerging Diseases

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A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of Tembusu virus (TMUV) infection. The reaction was performed in one step in a single tube at 64°C for 45 min, with SYBR Green I dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10 copies/μl, and no cross-reaction with other avian viruses was observed. The assay was evaluated further for the diagnosis of TMUV in field samples and compared with conventional RT-PCR, demonstrating that results of the RT-LAMP assay corresponded to those of conventional RT-PCR. In conclusion, RT-LAMP with SYBR Green I dye was shown to be a sensitive, simple assay for the rapid diagnosis of TMUV infection in ducks.

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Usefulness of RT-PCR for the diagnosis of Japanese encephalitis in clinical samples

Swami

Ratho

Mishra

et al. 2008

Scandinavian Journal of Infectious Diseases

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The present study was carried out between July 2003 and December 2005 in PGIMER, Chandigarh, India and aimed to compare IgM capture ELISA and nested RT-PCR for the diagnosis of Japanese encephalitis (JE). The samples collected were cerebrospinal fluid and blood from 40 febrile patients with encephalitis (n=40, group I) and blood samples from febrile patients without encephalitis residing in JE endemic areas (n=45, group II). Overall, in CSF samples JE specific RNA was detected in 9/40 (22.5%), while 7/28 (25%) patients showed the presence of specific IgM antibodies. Only 28 CSF samples could be subjected to both RT-PCR and IgM and, among these, 13 cases were found to be confirmed JE based on IgM and/or RT-PCR positivity. Among the confirmed cases, 6 (6/13, 46.5%) could be detected by RT-PCR alone, 4 (4/13, 30.7%) by IgM capture ELISA and 3 (3/13, 23.1%) patients were positive by both the methods. All the RT-PCR positive cases had presented within 5 d of onset of illness. The serum samples of only 16 patients in group I could be tested for IgM antibodies and 5 (31.25%) were found to be positive, while in group II, 11.1% (5/45) positivity was observed. JE specific RNA could not be detected in serum samples of either group of patients. This study highlights the need for carrying out RT-PCR in CSF samples, compared to IgM antibody detection, for the early detection of JEV.

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Application of One Step RT-PCR Assay for Detection of Flavivirus RNA in Mosquitoes

Cited by 4 publications

References 8 publications

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

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

Rapid Detection of Tembusu Virus by Reverse-Transcription, Loop-mediated Isothermal Amplification (RT-LAMP)

Usefulness of RT-PCR for the diagnosis of Japanese encephalitis in clinical samples

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