Identification of Entamoeba histolytica by FTA-nested PCR
LU Yan, CHEN Jia-xu, ZHANG Yong-nian, LI Hao, CHU Yan-hong, AI Lin, CAI Yu-chun, CHEN Shao-hong
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Key Laboratory of Parasite and Vector Biology, MOH; WHO Collaborating Center of Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China
Abstract：To establish a simple and rapid FTA-nested PCR method for identification of the Entamoeba histolytica (E.h), the fecal samples of diarrheal patients were collected. The samples were examined with a microscope firstly, and 44 positive samples were selected. The genomic DNA of Entamoeba in positive samples was extracted by FTA filters. Primers were designed based on the SSU rRNA fragment of E.h and the plate DNA was amplified by nested PCR. All PCR products were detected by agarose gel electrophoresis, and then the target fragments were sequenced and analyzed by BLAST. The 427 bp fragment of DNA was detected from 20 fecal samples. Sequence analysis of 20 target fragments amplified by nested PCR showed that they were E.h. The consistent rate of microscopic examination and nested PCR was 45.45% (20/44). This nested PCR could identify and differentiate the pathogenic E.h from the non- pathogenic Entamoeba species which were morphologically identical (similar). It suggested that FTA-nested PCR is a simple, rapid and reliable technique for identifying E.h in human stool samples, and provided a new technical method for differential diagnosis of E.h in clinical examination and epidemiological survey.
[1] World Health Organization. World Health Organization/Pan American Health Organization/UNESCO report of a consultation of experts on amoebiasis[J]. Wkly Epidemiol Rec, 1997, 72: 97-99. [2] Tanyuksel M,Petri WA Jr. Laboratory diagnosis of amebiasis[J]. Clin Microbiol Rev, 2003, 16(4): 713-729. [3] WHO/PAHO/UNESCO report. A consultation with experts on amoebiasis. Mexico City, Mexico 28-29 January, 1997[J]. Epidemiol Bull, 1997, 18(1): 13-14. [4] Sargeaunt PG. Entamoeba histolytica and E. dispar [J]. Trans R Soc Trop Med Hyg, 1994, 8(2): 254-255. [5] Ali IK, Clark CG, Petri WA Jr. Molecular epidemiology of amebiasis[J]. Infect Genet Evol, 2008, 8(5): 698-707. [6] Delialioglu N, Aslan G, Ozturk C, et al. Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey[J]. J Parasitol, 2008, 94(2): 530-532. [7] Zaki M, Meelu P, Sun W, et al. Simultaneous differentiation and typing of Entamoeba histolytica and Entamoeba dispar [J]. J Clin Microbiol, 2002, 40(4): 1271-1276. [8] Haque R, Petri WA Jr. Diagnosis of amebiasis in Bangladesh[J]. Arch Med Res, 2006, 37(2): 273-276. [9] Hamzah Z, Petmitr S, Mungthin M, et al. Differential detection of Entamoeba histolytica , Entamoeba dispar , and Entamoeba moshkovskii by a single-round PCR assay[J]. J Clin Microbiol, 2006, 44(9): 3196-3200. [10] Evangelopoulos A, Spanakos G, Patsoula E, et al. A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytic a and Entamoeba dispar in faeces[J]. Ann Trop Med Parasitol, 2000, 94(3): 233-240. [11] Santos FL, Gon alves Mde S, Soares NM. Validation and utilization of PCR for differential diagnosis and prevalence determination of Entamoeba histolytica / Entamoeba dispar in Salvador City, Brazil[J]. Braz J Infect Dis, 2011, 15(2): 119-125. [12] Stangegaard M, Borsting C, Ferrero-Miliani L, et al. Evaluation of four automated protocols for extraction of DNA from FTA cards[J]. J Lab Autom, 2013, 18(5): 404-410. [13] Kato H, Caceres AG, Mimori T, et al. Use of FTA cards for direct sampling of patients' lesions in the ecological study of cutaneous leishmaniasis[J]. J Clin Microbiol, 2010, 48(10): 3661-3665. [14] Nantavisai K, Mungthin M, Tan-ariya P, et al. Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalis in stool specimens[J]. J Clin Microbiol, 2007, 45(2): 581-583. [15] Zhang XP, Zhu Q, He YY, et al. Detection of Cryptospordium spp. in environmental water samples by FTA-PCR[J]. Chin J Schisto Ctrl, 2011, 23(1): 71-73. (in Chinese) 张小萍, 朱倩, 何艳燕, 等. FTA-PCR检测水源性隐孢子虫研究 [J].中国血吸虫病防治杂志, 2011, 23(1):71-73. [16] Tian LG, Zhang XP, Chen SH, et al. Detection of Cryptosporidium spp. In human stool samples with FTA-PCR[J]. Int J Med Parasit Dis, 2011, 38(4): 211-214. (in Chinese) 田利光, 张小萍, 陈韶红, 等. 应用FTA/PCR检测粪便中隐孢子虫的实验研究[J]. 国际医学寄生虫病杂志, 2011, 38(4):211-214. [17] Chen SH, Zhang YN, Li SQ, et al. Preliminary study on detection of metacercariae in aquatic products by FTA[J]. Chin J Zoonoses, 2010, 26(10): 931-934. (in Chinese) 陈韶红, 张永年, 李树清, 等. 应用FTA法检测水产品中吸虫囊蚴的初步研究[J]. 中国人兽共患病学报, 2010, 26(10):931-934. [18] Rodulfo H, Ahmar B, Rodriguez ME, et al. Nested PCR reveals elevated over-diagnosis of E. histolytica in Barcelona, Venezuela[J]. Invest Clin, 2012, 53(4): 365-377. [19] Ngui R, Angal L, Fakhrurrazi SA, et al. Differentiating Entamoeba histolytica , Entamoeba dispar and Entamoeba moshkovskii using nested polymerase chain reaction (PCR) in rural communities in Malaysia[J]. Parasit Vectors, 2012, 5: 187. [20] Liu H, Sun XD, Nie RH, et al. Comparison of the three methods used in detection of amoebiasis in feces[J]. Chin J Pathogen Biol, 2008, 3(9): 679-680, 689. (in Chinese) 刘慧, 孙晓东, 聂仁华, 等. 3种方法检测粪便中溶组织内阿米巴的结果比较[J]. 中国病原生物学杂志, 2008, 3(9):679-680, 689.
