Optimizing RT-semi-nested PCR Conditions with Newly Developed Primers for the Specific Detection of Human Enterovirus D68 and its Identification from Clinical Specimens

Human enterovirus D68 (EV-D68) is a member of the Enterovirus genus (EVs) within the Picornaviridae family, and generally associated with respiratory tract infection. Meanwhile, previous reports suggest that EV-D68 was also associated with acute flaccid paralysis in the clinical cases in USA and Japan. Two conventional PCR assays have been commonly used for genetic analysis of EVs. One is the RT-semi-nested PCR assay (VP4-VP2-PCR) that amplifies the VP4-VP2 region for detection of EVs with high sensitivity and the other is the CODEHOP RT-semi-nested PCR assay (CODEHOP PCR) that amplifies the VP1 region for typing. These assays are useful for detection and identification of EVs in many clinical cases. However, we experienced some cases in which we could not identify EV-D68 with CODEHOP PCR from clinical specimens in spite of the results that EV-D68 sequences were detected by VP4-VP2-PCR. In addition, these assays would have trouble in detecting target sequences in those cases where the specimens under examination have been acquired from patients infected with multiple types of EVs. Therefore, we tried to develop a novel RT-semi-nested PCR assay that has high sensitivity and specificity for EV-D68. We designed original primers to be used for the amplification of the VP1 region of EV-D68 and optimized the RT-semi-nested PCR conditions. Sensitivity and specificity were examined with previ-ously accumulated clinical specimens.