基于LAMP的人轮状病毒检测方法及其在水环境中的应用

LAMP based method for detecting human rotavirus and its application in water samples

环介导的恒温扩增(LAMP)是新兴的一种快速DNA扩增技术,具有特异性强、敏感性高、反应迅速、设备简单等特点,在人类病毒性疾病的诊断领域有广泛的应用。为快速、高效地检测人轮状病毒,根据其特异性的VP7基因,设计了一套特异性引物对该基因进行LAMP,同时优化其反应条件。结果表明,LAMP最佳反应条件为:镁离子终浓度3 mmol/L,甜菜碱终浓度1 mol/L的25μL体系,65℃反应90min。扩增终产物酶切分析得到的条带大小与预期结果(280bp、193bp和78 bp)相吻合,以人轮状病毒、星状病毒及诺如病毒为模板进行特异性测试没有发生交叉反应。将人轮状病毒质粒等倍稀释后分别用LAMP及PCR检测其灵敏度,分别达到5.08 copies/μL和5.08×102copies/μL。用优化的LAMP分别检测北京地区12个生活饮用水及12个再生水水样,其人轮状病毒检出率分别为16.7%(2/12)和25%(3/12)。研究表明,LAMP是一种程序简单、灵敏度和特异性较高的检测手段,在人轮状病毒的快速检测方面具有一定的开发潜力。

The present paper intends to introduce a method known as loop-mediated isothermal amplification, short for LAMP, for detecting rotavirus, a kind of toxic virus responsible for the acute viral gas- troenteritis in the infants and young children in the worldwide sphere. As is known, after replication in the gastrointestinal tract, rotaviruses are usually excreted in large amount and then disperse into the envi- ronment water. The stability of rotaviruses in the environmental water and their resistance to the physicoehemical treatment process is likely to facilitate their transmission through water. A lot of studies confirm the presence of rotaviruses in the sewage and even in the treated ef- fluents. To combat the spread and transmission of such viruses, a novel, rapid and sensitive technique called loop-mediated isothermal amplification（LAMP） has been developed in recent years, which at- tempts to use a DNA polymerase that is very active in stranding the displacement of DNA synthesis and a set of four specially designed primers that recognize a total of six distinct sequences on the targeted DNA. The LAMP method can operate in an extremely high speed. It is also cost-effective, sensitive and specific for the clinic service, and that is why it is now widely used in the area of virus detection. In the present paper, we have renovated the LAMP based-method for quick- finding human rotavirus. By means of the specific VP7-gene of human rotavirus, we have worked out a set of specific primers to amplify the special DNA sequence with the help of LAMP. In addition, we have managed to optimize the reaction conditions of LAMP. The experimen- tal data indicate that the optimal amplification condition proves to be at 65 ℃ for 90 min with 3 mmol/L magnesium ions and 1 mol/L Be- taine. The specificity of RT- LAMP assay has been further validated by the cross-reaction with different viruses （human rotavirus, human astrovirus and human norovirus） and the constrained analysis of the amplified products, from which we have attained three main bands （280 bp, 193 bp and 78 bp） . The sensitivity of the LAMP and PCR has been found equal to 5.08 copies/μL and 5.08 ×10^2 copies/μL, respectively. Thus, the renovated LAMP method can be successfully used for detecting human rotavirus in 12 samples of human drinking water and 12 reclaimed water. The detection rate of human rotavirus can be made as high as to 16.7 % （2/12） and 25% （3/12）, respec- tively. And, therefore, it can be concluded that all the results we have gained prove that LAMP serves as a simple, highly-sensitive and specific method for quick-detecting human rotavirus.