Detection of measles virus RNA on SYBR green real-time reverse transcription-polymerase chain reaction.

摘要

BACKGROUND: As the coverage rate of the measles vaccine increases, not all patients present the typical symptoms of measles after exposure to the measles virus (MV). The virus loads in clinical specimens from patients with vaccine-modified non-typical measles are expected to be low compared with those of primary MV infection. A rapid and sensitive laboratory procedure is required for diagnosis of measles. METHODS: SYBR Green (TaKaRa) and TaqMan (ABI) real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect MV-RNA. For the real-time RT-PCR, primer sets were designed from a region of the MV H gene of the Edmonston strain (genotype A). A TaqMan probe specific for the H gene of genotype D MV was used. The minimum detectable level of MV-RNA in the SYBR Green and TaqMan real-time RT-PCR assays was evaluated using synthetic MV-RNA. The sensitivity of real-time RT-PCR was compared with that of nested RT-PCR and the virus isolation method using throat swabs and peripheral blood samples from patients with measles. RESULTS: The minimum detectable level of RNA was 10 and 10(2) copies for SYBR Green RT-PCR and TaqMan RT-PCR, respectively. Ten-10(6) copies of standard RNA were linearly detected on SYBR Green RT-PCR. The sensitivity of SYBR Green RT-PCR was equal to that of nested RT-PCR. MV-RNA was detected in virus isolation-negative throat swabs on SYBR Green RT-PCR. CONCLUSION: SYBR Green RT-PCR is a highly sensitive, rapid, and useful diagnostic procedure for the detection of MV.