Development of a Transcription-Reverse Transcription Concerted Reaction Method for Specific Detection of Human Enterovirus 71 from Clinical Specimens

Abstract

A transcription-reverse transcription (RT) concerted reaction (TRCR) method was developed for rapid and specific detection of EV71 from clinical specimens. This method was validated with EV71 strains from all of the known genotypes (genotypes A, B1 to B5, and C1 to C5), with detection limits of 10 to 10 3 copies, and was useful for identification of EV71 from throat swabs of patients with hand, foot, and mouth disease (HFMD). Enterovirus 71 (EV71) is a major etiological agent for hand, foot, and mouth disease (HFMD) and herpangina, along with coxsackievirus A (CVA) types such as CVA10 and CVA16 (1, 10). In contrast to CVA infection (3), EV71 infection is also associated with severe neurological diseases, such as brain-stem encephalitis and poliomyelitis-like paralysis (4, 9, 14) with a high incidence (case severity rate of Ͻ0.3%) (6). Effective vaccines are not available at present, and only supportive care can be provided for the treatment of severe brain-stem encephalitis (reviewed in reference 15). Because of the rapid progression of symptoms in fatal cases of EV71 infection (Յ12 h after hospitalization) (2), rapid diagnosis of EV71 infection is essential for severely EV71-infected patients to provide proper care among patients with or without HFMD caused by other EV infections.In this study, we aimed to develop a diagnostic test for EV71 by using a transcription-reverse transcription (RT) concerted reaction (TRCR) method (7, 13). The TRCR method is an isothermal RNA amplification method as well as an RT-loop-mediated isothermal amplification (LAMP) method, but it utilizes T7 RNA polymerase and avian myeloblastosis virus (AMV) reverse transcriptase instead of DNA polymerase (7, 13). The major advantages of the TRCR method are (i) rapid detection (20 min of reaction time), (ii) high specificity (by intercalation activating fluorescence [INAF] probes) (8), and (iii) high sensitivity (as low as 10 copies). For its rapid measurement of RNA, the TRCR method has been used as a diagnostic tool for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare in some hospitals

Comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis

“…In previous studies, many authors have used different collection methods to identify EV (throat swab) [28], HMPV (nasal swab) [29], rhinovirus (nasal and throat swab) [30], influenza (throat and nasal swab) [31], and RSV (nasopharyngeal aspirate and nasal swab) [19]. Moreover, Moës et al used bronchoalveolar lavage, pharyngeal swabs, nasopharyngeal aspirates, and sputum samples for the identification of coronavirus, although the study did not aim to compare the efficacy of sampling methods [32].…”

The uncoating of EV71 in mature late endosomes requires CD-M6PR

“…The EV71 strains SK-EV006/Malaysia/97 (SK-EV006; genogroup B) ( Shimizu et al, 1999 ), EV71/86-11316 NETH-86 (genogroup B) ( Nakajima et al, 2012 ) and EV71-GFP (generously gifted by Dr. M. Arita), which expresses GFP upon viral replication ( Yamayoshi et al, 2013 ), were used in this study. EV71/86-11316 NETH-86 was used for producing light-sensitive virus, and EV71-GFP was used in siRNA knockdown experiment.…”

Diagnostic Approaches For COVID-19: Lessons Learned and the Path Forward

“…The test is fast, with 30 min turn-around time and is easy to use by a layperson; however, each test cost around $85 and is not economically feasible for mass use. 24 Other isothermal amplification techniques were also explored including the nicking enzyme amplification reaction (NEAR), 83 , 84 transcription-mediated amplification (TMA), 85 transcription reverse-transcription concerted reaction (TRC), 86 and smart amplification process (SmartAmp). 87 The NEAR amplifies DNA exponentially at a constant temperature from 55 to 59 °C.…”