虫卵ITS-2序列分析鉴别华支睾吸虫和扇棘单睾吸虫的研究
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摘要
目的建立以分子生物学技术为基础的华支睾吸虫与扇棘单睾吸虫的ITS-2序列分析鉴别方法。方法在华支睾吸虫流行区采集华支睾吸虫和扇棘单睾吸虫成虫,从成虫虫体分离并收集虫卵。以蛔虫、鞭虫、钩虫、蛲虫做对照虫种,按不同虫种和虫卵数分成5个实验组,各组分别提取DNA,并扩增ITS-2序列,对扩增产物进行测序并作同源性分析以确保为目标片段,根据PCR扩增产物电泳获得的条带判定虫种。结果以华支睾吸虫和扇棘单睾吸虫虫卵DNA为模板的PCR产物电泳图谱分别在400bp、500bp左右各显示一条明显条带;在以两种虫卵DNA混合液为模板的PCR产物中,电泳图谱在400bp左右和500bp左右各显示明显条带。将测序得到的两种核苷酸序列进行Blast比对后,显示与GenBank中相应的华支睾吸虫和扇棘单睾吸虫序列高度同源。混入四种肠道线虫虫卵DNA的PCR产物中,除目的条带外,无其他条带。结论该方法可以对华支睾吸虫及扇棘单睾吸虫虫卵进行鉴别,且结果不受常见四种肠道线虫虫卵干扰,敏感性和特异性较高,可作为两虫种的鉴别检测方法。
Objective To establish an identification method basis on molecular biology techniques for Clonorchis sinensis and Haplorchis taichui.Methods Adult flukes of C.sinensis and H.taichui were collected from Clonorchiasis endemic areas and the eggs were separated from the worms under stereomicroscope.Roundworm,Whipworm,Hookworm and Pinworm were served as control species,all kinds of eggs were divided into five experimental groups according to the species and the number of eggs.DNA from each group was extracted and amplified the sequence of ITS-2,and amplified products were sequenced and homology analyzed ensure that the products were the target fragments.The species of insect were determined according to the electrophoresis of PCR products.Results The electrophoresis of PCR products of C.sinensis and H.taichui display a distinct band at around 400bp,500bp,respectively; electropherogram in PCR products of mixture DNA of two species eggs showed two obvious bands at around400bp and 500bp.The two nucleotide sequencing which had sequenced were compared to Blast,show that both of the sequences are highly homologous with C.sinensis and H.taichi in GenBank,respectively.The PCR products with four kinds of Heligmosomoides polygyrus eggs have no other bands except the objective strips.Conclusion This method can be used to identify C.sinensis and H.taichui,and the results are not affected by four kinds of Heligmosomoides polygyrus eggs,is an identification method for Clonorchis sinensis and Haplorchis taichui with high sensitivity and specificity.
Objective To establish an identification method basis on molecular biology techniques for Clonorchis sinensis and Haplorchis taichui.Methods Adult flukes of C.sinensis and H.taichui were collected from Clonorchiasis endemic areas and the eggs were separated from the worms under stereomicroscope.Roundworm,Whipworm,Hookworm and Pinworm were served as control species,all kinds of eggs were divided into five experimental groups according to the species and the number of eggs.DNA from each group was extracted and amplified the sequence of ITS-2,and amplified products were sequenced and homology analyzed ensure that the products were the target fragments.The species of insect were determined according to the electrophoresis of PCR products.Results The electrophoresis of PCR products of C.sinensis and H.taichui display a distinct band at around 400bp,500bp,respectively; electropherogram in PCR products of mixture DNA of two species eggs showed two obvious bands at around400bp and 500bp.The two nucleotide sequencing which had sequenced were compared to Blast,show that both of the sequences are highly homologous with C.sinensis and H.taichi in GenBank,respectively.The PCR products with four kinds of Heligmosomoides polygyrus eggs have no other bands except the objective strips.Conclusion This method can be used to identify C.sinensis and H.taichui,and the results are not affected by four kinds of Heligmosomoides polygyrus eggs,is an identification method for Clonorchis sinensis and Haplorchis taichui with high sensitivity and specificity.
引文
[1]方怡悦,陈颖丹,黎学铭,等.我国华支睾吸虫病流行区感染现状调查[J].中国寄生虫学与寄生虫病杂志,2008,26(2):99-103,109.
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[3]杨益超,李树林,谭裕光,等.广西肝吸虫流行区人群及淡水鱼扇棘单睾吸虫感染调查[J].应用预防医学,2012,18(2):75-77.
[4]李艳文,刘小泉,何登贤,等.血清学和B超声像法检查华支睾吸虫感染比较[J].应用预防医学,2010,16(2):107.
[5]Dvorak J,Vanacova S,Hampl V,et al.Comparison of European Trichobilharzia species based on ITS1 and ITS2 sequences[J].Parasitology,2002,124(Pt 3):307-313.
[6]Iwagami M,Monroy C,Rosas MA,et al.A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador[J].J Helminthol,2003,77(1):33-38.
[7]熊天擎,杨斌斌,程瑜静,等.虫卵基因序列分析鉴定斯氏并殖吸虫病[J].热带医学杂志,2011,11(11):1248-1251,封3.
[8]Megumi Sato,Urusa Thaenkham,et al.Discrimination of O.viverrini,C.sinensis,H.pumilio and H.taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions[J].J Acta Tropica,2009,109(1):81-83.
[9]Le TH,Van De N,Blair D,et al.Clonorchis sinensis and Opisthorchis viverrini:development of a mitochondrialbased multiplex PCR for their identification and discrimination[J].Exp Parasitol,2006,112(2):109-114.
[10]Thaenkham U,Visetsuk K,Dung do T,et al.Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker[J].Acta Trop,2007,103(1):26-32.
[11]Wongratanacheewin S,Pumidonming W,Sermswan RW,et al.Detection of Opisthorchis viverrini in human stool specimens by PCR[J].J Clin Microbiol,2002,40(10):3879-3880.
[12]Parvathi A,Sanath Kumar H,Kenchanna Prakasha B,et al.Clonorchis sinensis:development and evaluation of a nested polymerase chain reaction(PCR)assay[J].Exp Parasitol,2007,115(3):291-295.
[13]Leonore lovis,Tippi K.Mak,Khampheng Phongluxa,et al.PCR diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in a lao Community in an area of endemicity and comparison of diagnostic methods for parasitological field surveys[J].Journal of clinical microbiology,2009,47(5):1517-1523.
[14]Jong-Yil Chai,Tai-Soon Yong,Keeseon S.Eom,et al.Hyperendemicity of Haplorchis taichui infection among riparian People in Saravane and Champasak Province,lao PDR[J].Korean J Parasitol,2013,51(3):305-311.
[15]Rebecca J.Traub,Julie Macaranas,Mathirut Mungthin,et al.A new PCR-based approach indicates the range of Clonorchis sinensis now extends to central Thailand[J].PloS Negl Trop Dis,2009,3(1):367.
[2]黎学铭,杨益超,蓝春庚,等.广西发现扇棘单睾吸虫[J].中国寄生虫学与寄生虫病杂志,2004,22(1):61.
[3]杨益超,李树林,谭裕光,等.广西肝吸虫流行区人群及淡水鱼扇棘单睾吸虫感染调查[J].应用预防医学,2012,18(2):75-77.
[4]李艳文,刘小泉,何登贤,等.血清学和B超声像法检查华支睾吸虫感染比较[J].应用预防医学,2010,16(2):107.
[5]Dvorak J,Vanacova S,Hampl V,et al.Comparison of European Trichobilharzia species based on ITS1 and ITS2 sequences[J].Parasitology,2002,124(Pt 3):307-313.
[6]Iwagami M,Monroy C,Rosas MA,et al.A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador[J].J Helminthol,2003,77(1):33-38.
[7]熊天擎,杨斌斌,程瑜静,等.虫卵基因序列分析鉴定斯氏并殖吸虫病[J].热带医学杂志,2011,11(11):1248-1251,封3.
[8]Megumi Sato,Urusa Thaenkham,et al.Discrimination of O.viverrini,C.sinensis,H.pumilio and H.taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions[J].J Acta Tropica,2009,109(1):81-83.
[9]Le TH,Van De N,Blair D,et al.Clonorchis sinensis and Opisthorchis viverrini:development of a mitochondrialbased multiplex PCR for their identification and discrimination[J].Exp Parasitol,2006,112(2):109-114.
[10]Thaenkham U,Visetsuk K,Dung do T,et al.Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker[J].Acta Trop,2007,103(1):26-32.
[11]Wongratanacheewin S,Pumidonming W,Sermswan RW,et al.Detection of Opisthorchis viverrini in human stool specimens by PCR[J].J Clin Microbiol,2002,40(10):3879-3880.
[12]Parvathi A,Sanath Kumar H,Kenchanna Prakasha B,et al.Clonorchis sinensis:development and evaluation of a nested polymerase chain reaction(PCR)assay[J].Exp Parasitol,2007,115(3):291-295.
[13]Leonore lovis,Tippi K.Mak,Khampheng Phongluxa,et al.PCR diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in a lao Community in an area of endemicity and comparison of diagnostic methods for parasitological field surveys[J].Journal of clinical microbiology,2009,47(5):1517-1523.
[14]Jong-Yil Chai,Tai-Soon Yong,Keeseon S.Eom,et al.Hyperendemicity of Haplorchis taichui infection among riparian People in Saravane and Champasak Province,lao PDR[J].Korean J Parasitol,2013,51(3):305-311.
[15]Rebecca J.Traub,Julie Macaranas,Mathirut Mungthin,et al.A new PCR-based approach indicates the range of Clonorchis sinensis now extends to central Thailand[J].PloS Negl Trop Dis,2009,3(1):367.