收稿日期: 2021-02-02
修回日期: 2021-03-14
网络出版日期: 2021-07-05
基金资助
国家重点研发计划课题(2018YFC1602504);国家重点研发计划课题(2017YFC1601200);国家自然科学基金(31560655);国家自然科学基金(31960707)
Establishment of a Giardia lamblia detection method based on the LAMP microfluidic chip
Received date: 2021-02-02
Revised date: 2021-03-14
Online published: 2021-07-05
Supported by
Supported by National Key Program Projects(2018YFC1602504);Supported by National Key Program Projects(2017YFC1601200);National Natural Science Foundation of China(31560655);National Natural Science Foundation of China(31960707)
选择蓝氏贾第鞭毛虫（简称贾第虫）磷酸丙糖异构酶（TPI）基因作为靶基因设计引物,以包含TPI基因序列的人工合成质粒为模板,建立基于介导等温扩增（LAMP）微流控芯片的贾第虫检测方法,并对该方法的特异性、敏感性、重复性和稳定性进行评价。结果显示,建立的贾第虫LAMP微流控芯片检测方法可在30 min内完成核酸的扩增,检测敏感性可达4.0 × 10拷贝/μl和0.01 ng核酸/μl。隐孢子虫、旋毛虫、艾美耳球虫、戊肝病毒和粪肠球菌核酸均不能扩增,对贾第虫具有良好的特异性。以不同稀释浓度重组质粒为模板进行稳定性和重复性实验,结果显示核酸浓度与反应时间具有良好的相关性,对阳性粪样的检测结果显示扩增信号。建立了基于LAMP微流控芯片的贾第虫核酸检测方法,该方法操作简单、特异性强。
于铭川 , 杨忠委 , 王华然 , 师丹阳 , 周树青 , 尹静 , 孙树民 . 基于LAMP微流控芯片检测蓝氏贾第鞭毛虫方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2021 , 39(3) : 402 -405 . DOI: 10.12140/j.issn.1000-7423.2021.03.020
The triose phosphate isomerase (TPI) gene of Giardia lamblia was selected as the target gene, for which primers were designed, and the synthetic plasmid containing TPI gene sequence was used as the template to establish a LAMP microfluidic chip detection method for detection ofG. lamblia. The specificity, sensitivity, repeatability and stability of the method were evaluated. The results showed that the established LAMP microfluidic chip detection method for G. lamblia could complete nucleic acid amplification within 30 min, and the detection sensitivity reached 4.0 × 10 copies/μl and 0.01 ng nucleic acid/μl. In addition, this method showed high specificity for G. lamblia, while not being able to amplify the nucleic acids of Cryptosporidium, Trichinella spiralis, Eimeria, hepatitis E virus or Enterococcus faecalis. The stability and repeatability of the method were tested using different dilutions of recombinant plasmids. The results showed that the nucleic acid concentration was highly correlated with the reaction time, and positive fecal samples presented amplifying signals. The results demonstrate the establishment of a lamp microfluidic chip-based nucleic acid detection method forG. lamblia. The method is simple and highly specific.
Key words： Giardia lamblia; Microfluidic chip; LAMP; TPI gene
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