Okafuji Takao, Yoshida Naoko, Fujino Motoko, Motegi Yoshie, Ihara Toshiaki, Ota Yoshinori, Notomi Tsugunori, Nakayama Tetsuo
Okafuji Pediatric Clinic, Himeji, Hyougo, Japan.
J Clin Microbiol. 2005 Apr;43(4):1625-31. doi: 10.1128/JCM.43.4.1625-1631.2005.
Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.
大多数腮腺炎患者无需任何病毒学检查即可临床诊断，但部分诊断为腮腺炎的病例可能由其他病原体或疫苗接种失败（SVF）继发引起。为阐明这些问题，需要一种灵敏、特异且快速的诊断方法。我们从34例自然感染患者病程中获取了60份唾液拭子、从疫苗相关腮腺炎患者中获取了10份样本以及从疫苗接种失败患者中获取了5份样本。提取总RNA并进行逆转录聚合酶链反应（RT-PCR）和环介导等温扩增（LAMP）以进行基因组扩增。我们通过LAMP在RNA提取后60分钟内检测到相当于0.1个蚀斑形成单位（PFU）的腮腺炎病毒RNA，其灵敏度与RT巢式PCR相同。33份样本中有30份在第2天内分离出腮腺炎病毒，其中32份通过LAMP扩增出腮腺炎病毒基因组。通过监测达到浊度阈值的时间来计算病毒滴度。病毒载量在第3天后下降，在血清学诊断为疫苗接种失败且病情较轻的患者中较低。LAMP检测腮腺炎病毒基因组的准确性得到了证实；此外，它有助于计算反映疾病发病机制的病毒载量。
