Detection of amount of protoscolices of Echinococcus granulosus with resazurin reduction method
RUOSHANGULI Rou-zi1, 2, GAO Hui-jing1, LIU Hui1, XIAO Yun-feng1, YANG Ning1, ZHAO Jun1, WANG Jian-hua1, LIN Ren-yong1, WEN Hao1, LYU Guo-dong1
1.Xinjiang Major Disease Medical Laboratory and Key Laboratory of the Ministry and Province Cultivation Base,Clinical Medical Research Institute, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China; 2.Xinjiang Bestmind Biological Technology Development Co,. Ltd., Urumqi 830054, China
Abstract:We aimed to establish a new experimental method to detect the total number of living protoscolices of Echinococcus granulosus based on the reduction characteristic of resazurin that the resazurin could emit strong fluorescence when reduced by mitochondrial enzyme. The fluorescence values of the protoscolices stained with resazurin was determined by fluorescence spectrophotometer (excitation wavelength at 560 nm, emission wavelength 590 nm), and the morphology were observed by laser scanning confocal microscope. A mathematical model was established based on the relationship between the number of living protoscolices and the corresponding fluorescence values through the statistical method. Results of the laser scanning confocal microscope showed that the living protoscolices stained by resazurin emitted red fluorescence while the death one emitted little fluorescence. According to the relationship between the measured fluorescence values and corresponding protoscolices number, a linear equation y=0.019x+6.453 was established, and the total numbers of living protoscolices could be obtained by putting the fluorescence value (y) into the formula. The correlation coefficient of the mathematical model was r=0.994 and P<0.01, which showed that the correlation coefficient r between living protoscolices number in each concentration and corresponding fluorescence values of each concentration was very significant. This study established a rapidly experimental method, which based on the reduction characteristic of resazurin, to detect the total number of living protoscolices of Echinococcus granulosus in vitro. This method avoids artificial errors and reduce the human consumption, and could be applied to screening anti-echinococcosis drugs in large scale in vitro.
[1] Zhang RN, Wulamu MMT. Recent advances on development of corproantigen detection system for dogs infected with Echinococcus spp[J]. Chin J Zoonoses, 2013, 04: 398-402. (in Chinese) 张瑞妮, 吾拉木·马木提. 棘球绦虫感染犬粪抗原检测研究新进展[J]. 中国人兽共患病学报, 2013, 04: 398-402. [2] Shi DZH, Analysis of China cystic enhinococcosis geography[J]. Endem Dis Bull, 2000, 15(1): 74-75. (in Chinese) 史大中. 中国囊性包虫病的地理分析[J]. 地方病通报, 2000, 15(1): 74-75. [3] Wang LY, Wu WP, Zhu XH. The endemic states of hydatidosis in China from 2004 to 2008[J]. Chin J Zoonoses, 2010, 26(7): 699-702. (in Chinese) 王立英, 伍卫平, 朱雪花. 2004-2008年全国包虫病疫情分析[J]. 中国人兽共患病学报, 2010, 26(7): 699-702. [4] Davis A, Pawlowski ZS, Dixon H. Multicentre clinical trials of benzimidazole carbamates in human echinococcosis[J]. Bull World Health Organ, 1986, 64(3): 383-388. [5] Davis A, Dixon H, Pawlowski ZS. Muhicentre clinical trials of benzimidazole carbarmates in human cystic echinococcosis (phase II)[J]. Bull World Health Organ, 1989, 67(5): 503-508. [6] Jiang CP. Hydotid diseases[M]. Beijing: Taihai Publishing House, 2001, (4): 1-2. (in Chinese) 蒋次鹏.包虫病学[M]. 北京: 台海出版社, 2001, (4): 1-2. [7] Guidelines for treatment of cystic and alveolar echinococcosis in humans. WHO Informal Working Group on Echinococcosis[J]. Bull World Health Organ, 1996, 74(3): 231-242. [8] Liu N, Su FW. Study on rapid detection of total bacteriacount in milk by using resazurin reduction method[J]. J Henan Univ Technol, 2013, 34(1): 73-76. (in Chinese) 刘娜, 苏丰薇.刃天青还原法快速检测鲜牛奶中总菌数 [J]. 河南工业大学学报:自然科学版, 2013, 34(1): 73-76. [9] Van den Driessche F, Rigole P, Brackman G, et al. Optimization of resazurin-based viability staining for quantification of microbial biofilms[J]. Microbiol Methods, 2014, 98: 31-34. doi:10.1016/j.mimet.2013.12.011 [10] Martin LM, Crenshaw C, Dean AJ. Determination of the number of motile sperm within an ovine semen sample using resazurin[J]. Small Ruminant Res, 1999, 2(36): 161-165. doi:10.1016/S0921-4488(98)00180-1 [11] Min TG, Woo SK. A simple quick and nondestructive method for brassicaceae seed viability measurement with single seed base using resazurin[J]. Hort Environ Biotechnol, 2011, 52(3): 240-245. [12] Mueller D, Tascher G, Damm G, et al. Real-time in situ viability assessment in a 3D bioreactor with liver cells using resazurin assay[J]. Cytotechnology, 2013, 65(2): 297-305. doi:10.1007/s10616-012-9486-6 [13] Schonfelder J, Valtink M, Knels L, et al. Quality assessment of corneal storage media and their components[J]. Graefes Arch Clin Exp Ophthalmol, 2014, 252(1): 77-82. doi:10.1007/s00417-013-2482-5 [14] Yuan LY, Zhang ZZ, Shi BX, et al. In vitro cultivation protoscoleces of the protoslices of Echinococcus granulosus in edium RPMI-1640 and MEM[J]. J Anim Sci Vet Med, 2008, 05: 16-18. (in Chinese) 袁丽英, 张壮志, 石保新, 等. 细粒棘球绦虫——原头蚴在两种细胞培养液中体外培养的初步观察[J]. 畜牧兽医杂志, 2008, 05: 16-18. [15] Mi XY, Gunuer TEX, Zhang ZZ, et al. Study on in vitro cultivation for protoscoleces of the Echinococcus multilocalaris [J]. Chin J Anim Infect Dis, 2011, 19(4): 67-70. (in Chinese) 米晓云, 古努尔.吐尔逊, 张壮志, 等. 多房棘球绦虫一泡球蚴的体外培养研究[J]. 中国动物传染病学报, 2011, 19(4): 67-70. [16] Samreen M, Margaret P. Effects of multivitamins and known teratogens on chick cardiomyocytes micromass culture assay[J]. Iran J Basic Med Sci, 2013, 16(9): 996-1003. [17] Shabafrooz V, Mozafari M, Kohler GA, et al. The effect of hyaluronic acid on biofunctionality of gelatin-collagen intestine tissue engineering scaffolds[J]. J Biomed Mater Res A, 2013, 10: 101-102. doi:10.1002/jbm.a.34984 [18] Mariscal A, Lopez-Gigosos RM, Carnero-Varo M, et al. Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm[J]. Appl Microbiol Biotechnol, 2009, 82(4): 773-783. doi:10.1007/s00253-009-1879-x [19] O'Brien J, Wilson I, Orton T, et al. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity[J]. Eur J Biochem, 2000, 267(17): 5421-5426.