Establishment of a Giardia lamblia detection method based on the LAMP microfluidic chip

doi:10.12140/j.issn.1000-7423.2021.03.020

Abstract

Abstract:

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

CLC Number:

R382.213

YU Ming-chuan, YANG Zhong-wei, WANG Hua-ran, SHI Dan-yang, ZHOU Shu-qing, YIN Jing, SUN Shu-min. Establishment of a Giardia lamblia detection method based on the LAMP microfluidic chip[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2021, 39(3): 402-405.

Figures/Tables 2

References 20

[1]	Dixon BR. Giardia duodenalis in humans and animals- transmission and disease[J]. Res Vet Sci, 2021,135:283-289. doi: 10.1016/j.rvsc.2020.09.034
[2]	Liu H, Xu N, Shen YJ, et al. Infection and genotype of Giardia lamblia among HIV/AIDS patients in Guangxi[J]. Chin J Parasitol Parasit Dis, 2019,37(3):321-325. (in Chinese)
	( 刘华, 徐宁, 沈玉娟, 等. 广西地区艾滋病病毒感染/艾滋病患者蓝氏贾第鞭毛虫感染情况及基因型分析[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(3):321-325.)
[3]	Yu YF, Wu XP, Chu YH, et al. Infection of Giardia lamblia in HIV-infected individuals and in kindergarden children in rural area of Anhui and genotype analysis[J]. Chin J Parasitol Parasit Dis, 2016,34(6):537-541. (in Chinese)
	( 俞英昉, 吴秀萍, 储言红, 等. 安徽农村地区HIV感染者和幼儿园儿童蓝氏贾第鞭毛虫感染情况及其基因型[J]. 中国寄生虫学与寄生虫病杂志, 2016,34(6):537-541.)
[4]	Teng XJ, Chen JX, Tian LG. Epidemic status of HIV/AIDS with intestinal protozoa infection[J]. Chin J Parasitol Parasit Dis, 2017,35(6):607-614. (in Chinese)
	( 滕雪娇, 陈家旭, 田利光. HIV/AIDS患者合并肠道原虫感染状况[J]. 中国寄生虫学与寄生虫病杂志, 2017,35(6):607-614.)
[5]	Han MY, An W, Ma JF, et al. Research process on the domestic prevalence status of zoonotic Cryptosporidium and Giardia[J]. J Pathog Biol, 2019,14(5):614-622. (in Chinese)
	( 韩明毅, 安伟, 马金锋, 等. 人畜共患贾第鞭毛虫和隐孢子虫国内研究进展[J]. 中国病原生物学杂志, 2019,14(5):614-622.)
[6]	Liu Y, Li SH, Zhang YL, et al. Analysis of current status of human intestinal protozoal infection in Henan Province[J]. Chin J Parasitol Parasit Dis, 2018,36(3):280-285. (in Chinese)
	( 刘颖, 李素华, 张雅兰, 等. 河南省人体肠道原虫感染现状分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(3):280-285.)
[7]	Yuan YX, Zheng GX, Wang DD, et al. Cloning and expression of the nucleoside diphosphate kinase gene of Giardia lamblia[J]. Chin J Parasitol Parasit Dis, 2018,36(2):190-192, 195. (in Chinese)
	( 袁义雪, 郑国侠, 王丹丹, 等. 蓝氏贾第鞭毛虫核苷二磷酸激酶基因的克隆与表达[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(2):190-192, 195.)
[8]	Kifleyohannes T, Robertson LJ. Preliminary insights regarding water as a transmission vehicle forCryptosporidium and Giardia in Tigray, Ethiopia[J]. Food Waterborne Parasitol, 2020,19:e00073.
[9]	Peng JJ, Zou Y, Li ZX, et al. Prevalence and multilocus genotyping of Giardia duodenalis in Tan sheep (Ovis aries) in northwestern China[J]. Parasitol Int, 2020,77:102126. doi: 10.1016/j.parint.2020.102126
[10]	Elsafi SH, Al-Maqati TN, Hussein MI, et al. Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum[J]. Parasitol Res, 2013,112(4):1641-1646. doi: 10.1007/s00436-013-3319-1
[11]	Wei XH, Chen SY. Advances in Epidemiology of Giardia lamblia and Cryptosporidium[J]. J Trop Med, 2014,14(2):265-268.
[12]	Zhang LS, Wang YJ, Cao JP. Progress and application of immunodiagnostic methods of giardiasis[J]. Chin J Schisto Control, 2017,29(3):385-387, 392. (in Chinese)
	( 章乐生, 王燕娟, 曹建平. 贾第虫病免疫学诊断方法及其应用研究进展[J]. 中国血吸虫病防治杂志, 2017,29(3):385-387, 392.)
[13]	Li J, Huang DN, Zhang XM, et al. Establishment of a two-step duplex fluorescence quantitative PCR method for simultaneous detection of Giardia lamblia and Cryptosporidium parvum[J]. Chin J Zoonoses, 2020,36(1):32-39. (in Chinese)
	( 李佳, 黄达娜, 张晓敏, 等. 蓝氏贾第鞭毛虫和微小隐孢子虫双重荧光定量PCR两步法检测技术的建立[J]. 中国人兽共患病学报, 2020,36(1):32-39.)
[14]	Liu YW. Establishment and application of loop-mediated isothermal amplification for detetion of Giardia SPP[D]. Nanjing: Nanjing Agricultural University, 2014. (in Chinese)
	( 刘雅文. 贾第鞭毛虫LAMP检测方法的建立与应用[D]. 南京: 南京农业大学, 2014.)
[15]	Zhang N, Qian YK, Wei S, et al. Based on recombinase polymerase amplification, the method of detection of grapevine leafroll-associated virus 3[J]. Xinjiang Agric Sci, 2016,53(2):302-308. (in Chinese)
	( 张娜, 乾义柯, 魏霜, 等. 基于重组酶聚合酶扩增技术(RPA) 的葡萄卷叶伴随病毒3号检测方法[J]. 新疆农业科学, 2016,53(2):302-308.)
[16]	Zhao Y, Wang Z, Jia SL, et al. Research progress of rapid detection of pathogens on microfluidic chip based on LAMP[J]. Chin J Lab Diagn, 2020,24(4):701-704. (in Chinese)
	( 赵阳, 王喆, 贾舒麟, 等. 基于LAMP的微流控芯片病原体快速检测方法研究进展[J]. 中国实验诊断学, 2020,24(4):701-704.)
[17]	Cui ZH, Wang LY, Cao LT, et al. Genetic characteristics and geographic segregation of Giardia duodenalis in dairy cattle from Guangdong Province, Southern China[J]. Infect Genet Evol, 2018,66:95-100. doi: 10.1016/j.meegid.2018.09.019
[18]	Yang XT. Establishment of ELISA, nested PCR, and LAMP detection assays for Giardia duodenalis[D]. Harbin: Northeast Agricultural University, 2019. (in Chinese)
	( 杨萱桐. 十二指肠贾第虫ELISA、巢式PCR和LAMP诊断方法的建立[D]. 哈尔滨: 东北农业大学, 2019.)
[19]	Shin JH, Lee SE, Kim TS, et al. Development of molecular diagnosis using multiplex real-time PCR and T4 phage internal control to simultaneously detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from human stool samples[J]. Korean J Parasitol, 2018,56(5):419-427. doi: 10.3347/kjp.2018.56.5.419
[20]	Ni BX, Liu YH, Xu XZ, et al. Establishment and evaluation of a novel DNA detection method based on recombinase-aided isothermal amplification assay for Giardia lamblia[J]. Chin J Schistoso Control, 2020,32(4):345-349. (in Chinese)
	( 倪碧娴, 刘燕红, 徐祥珍, 等. 重组酶介导的蓝氏贾第鞭毛虫特异性等温核酸扩增方法的建立及评价[J]. 中国血吸虫病防治杂志, 2020,32(4):345-349.)

引物名称	序列（5’→3’）
F3	GTGGACGTCGTCATTGCC
B3	TGCGTCTTTCAGAGTGCC
F1P	CTGCGCTGCTATCCTCAACTGTCGCCGTACACCTGTCAAC
B1P	TGTGTACCTAGAGGGGAACGGGCCCATGTCCTGAAGCATCTC