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