Ito Masahiro, Suga Tomoko, Akiyoshi Kyoko, Nukuzuma Souichi, Kon-no Mayumi, Umegaki Yasuhiro, Kohdera Urara, Ihara Toshiaki
Kyoto City Institute of Health and Environment Science, Nakagyo, Kyoto, Japan.
Pediatr Int. 2010 Aug;52(4):611-5. doi: 10.1111/j.1442-200X.2010.03124.x.
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.
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.
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.
SYBR Green RT-PCR is a highly sensitive, rapid, and useful diagnostic procedure for the detection of MV.
随着麻疹疫苗接种率的提高，并非所有接触麻疹病毒（MV）的患者都会出现典型的麻疹症状。与原发性MV感染患者相比，疫苗修饰的非典型麻疹患者临床标本中的病毒载量预计较低。麻疹的诊断需要一种快速且灵敏的实验室检测方法。
开发了SYBR Green（宝生物）和TaqMan（应用生物系统公司）实时逆转录聚合酶链反应（RT-PCR）检测法来检测MV-RNA。对于实时RT-PCR，从Edmonston株（基因型A）的MV H基因区域设计引物组。使用了一种针对基因型D MV的H基因的TaqMan探针。使用合成的MV-RNA评估SYBR Green和TaqMan实时RT-PCR检测法中MV-RNA的最低检测水平。将实时RT-PCR的灵敏度与巢式RT-PCR以及使用麻疹患者的咽拭子和外周血样本的病毒分离方法的灵敏度进行比较。
SYBR Green RT-PCR和TaqMan RT-PCR的RNA最低检测水平分别为10和10²拷贝。在SYBR Green RT-PCR上线性检测到10 - 10⁶拷贝的标准RNA。SYBR Green RT-PCR的灵敏度与巢式RT-PCR相同。在SYBR Green RT-PCR上，在病毒分离阴性的咽拭子中检测到了MV-RNA。
SYBR Green RT-PCR是一种用于检测MV的高度灵敏、快速且有用的诊断方法。
