Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification
Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification
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DOI：
10.1128/jcm.43.4.1625-1631.2005
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发表时间：
2005-04-01
影响因子：
9.4
通讯作者：
Nakayama, T
中科院分区：
文献类型：
--
作者：
Okafuji, T;Yoshida, N;Nakayama, T
大多数腮腺炎患者在临床诊断时没有任何病毒学检查，但一些确诊的腮腺炎病例可能是由其他病原体或二次疫苗失败（SVF）引起的。为了澄清这些问题，需要一种灵敏、特异和快速的诊断方法。我们从34例自然感染的患者中获得了60份唾液拭子，其中10份来自疫苗相关性腮腺炎患者，5份来自SVF患者。提取总RNA并进行逆转录-PCR（RT-PCR）和环介导等温扩增（LAMP）用于基因组扩增。在提取腮腺炎病毒RNA后的60 min内，我们用LAMP检测了相当于0.1 PFU的腮腺炎病毒RNA，其灵敏度与RT-nested PCR相同。33例腮腺炎病毒感染者中30例在感染后第2天内分离到腮腺炎病毒，其中32例经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的数量。病毒载量在第3天后下降，并且在血清学诊断为患有SVF且病情较轻的患者中较低。验证了LAMP检测腮腺炎病毒基因组的准确性，并有助于计算病毒载量，从而反映疾病的发病机制。
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.