基于环形泰勒虫Tams1基因多态性的二温式-PCR检测方法的建立

doi:10.3864/j.issn.0578-1752.2012.20.020

Abstract

Abstract: 【Objective】 It has been widespread concern for gene polymorphism assays in species classification, pathogen detection and vaccine screening. T. annulata is an important blood protozoa in bovine and causes major hazards. In present study the Tams1 amino acid polymorphism of T. annulata was analyzed and a two-temperature PCR detection method was established for T. annulata. 【Method】The specific primers were designed of Tams1 gene of T. annulata. A nucleotide fragment of 846 bp in length was obtained by PCR amplification. The gene amino acid sequence was compared and analyzed with 12 known species in different regions of the isolates in GenBank. And other primers were designed in conserved region of Tams1 gene and the detection method of T. annulata was established by two-temperature PCR. The method was used to detect theileriosis in field.【Result】The fragment encoded 281 amino acids, including 48 basic amino acids, 42 acidic amino acids and 100 hydrophobic amino acids. Identity analysis showed that Gansu strain of T. annulata had closest relationship with Ankara (Z48739.1), Turkey (AF214911), Bahrain (AF214794) and had little relationship with Xinjiang strain (No. accession number) while had closest relationship with Italy (AF214862) and Spain (AF214815) strains. The results can be testified by sequences alignment. The two-temperature PCR detection method can detect in 0.31 fg•μL-1 blood infection. Specificity results showed that only T. annulata genome DNA test was positive, other control parasite genome template were shown to be negative, and no cross reaction with other infected bovine Piroplasmorida. The two-temperature PCR method of T. annulata was used to test 335 field samples, the positive rate was 16.33%, and the microscopic detection results of 2.25% coincidence rate was 100%. Compared with common PCR, the method had no significant difference in sensitivity. But, two-temperature PCR was higher than common PCR in specificity, and the method took a short time with repeated heating and cooling.【Conclusion】T. annulata Tams1 genes from different strains exist obvious differences. Its amino acid sequence polymorphism feature is significant. Therefore, when the gene is used as a potential candidate antigen attention should be paid to the design of peptide vaccines. Two-temperature PCR method has good sensitivity and specificity. It has significance for early detection, early prevention of T. annulata.

Key words: Theileria annulata, Tams1, polymorphism, two-temperature PCR, detection method

LUO Jin, LIU Guang-Yuan, TIAN Zhan-Cheng, XIE Jun-Ren. Polymorphism Assays of Amino Acid and Establishment of a Two-Temperature PCR for Theileria Annulata Based on Tams1 Gene[J].Scientia Agricultura Sinica, 2012, 45(20): 4300-4309.

References

[1]Sutton A J, Karagenc T, Bakirci S, Sarali H, Pekel G, Medley G F. Modelling the transmission dynamics of Theileria annulata: model structure and validation for the Turkish context. Parasitology, 2012, 139(4): 441-453.

[2]Luo J X, Lu W S. Cattle Theileriosis in China. Tropical Animal Health and Production, 1997, 29(S4): 4-7.

[3]简子健, 黄家雨, 马素贞, 袁江玲, 苏贵成, 苗中秋, 沈炯玉, 张兰江, 李晓军. 新疆牛环形泰勒虫Tams1 基因的克隆与表达. 中国预防兽医学报, 2008, 30(10):784-789.

Jian Z J, Huang J Y, Ma S Z, Yuan J L, Su G C, Miao Z Q, Shen J Y, Zhang L J, LI X J. Cloning and expression of Tams1 gene from the bovine Theileria annulata in Xinjiang. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(10):784-789. (in Chinese)

[4]De Waal D T. The transovarial transmission of Babesia caballi by Hyalomma truncatum. Onderstepoort. Veterinary Research, 1990, 57: 99-100.

[5]董倩, 黄国强, 叶冰莹, 陈由强, 陈如凯. 二温式-PCR 在特异短核酸探针合成中的应用. 花生学报, 2008, 37(3):5-10.

Dong Q, Huang G Q, Ye B Y, Chen Y Q, Chen R K. Synthesis of specific and short RNA probes with two-temperature PCR. Journal of Peanut Science, 2008, 37(3):5-10. (in Chinese)

[6]陈亚利, 王卫萍, 周枚. 双温聚合酶链反应检测EB 病毒. 金陵医院学报, 1994 , 7 (3):237-240.

Chen Y L, Wang W P, Zhou M. Detection of Epstein-Barr virus DNA by two-temperature PCR cycles. Bulletin of Jinling Hospital, 1994, 7(3): 237-240. (in Chinese)

[7]Glascodine J, Tetley L, Tait A, Brown CGD, Shiels BR. Developmental expression of a Theileria annulata merozoite   surface antigen. Molecular and Biochemical Parasitology, 1990, 40: 105-112.

[8]Dickson J, Shiels B R. Antigenic diversity of a major merozoite surface molecule in Theileria annulata. Molecular and Biochemical Parasitology, 1993, 102: 305-311.

[9]Shiels B R, d’Oliveira C, McKellar S, Ben-Miled L, Kawazu S, Hide G. Selection of diversity at putative glycosylation sites in the immunodominant merozoite: piroplasm surface antigen of Theileria parasites. Molecular and Biochemical Parasitology, 1995, 72: 149-162.

[10]Katzer F, McKellar S, Ben Miled L, d’Oliveira C, Shiels B. Selection for antigenic diversity of Tams1; the major merozoite antigen of Theileria annulata. Annals of the New York Academy of Sciences, 1998, 849: 96-108.

[11]Gubbels M J, d'Oliveira C, Jongejan F. Development of an indirect Tams1 enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle. Clinical and Vaccine Immunology, 2000, 7(3):404-411.

[12]d’Oliveira C, Feenstra A, Vos H, Osterhaus A D M E, Shiels B     R, Cornelissen A W C A, Jongejan F. Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems. Vaccine, 1997, 15: 1796-804.

[13]Altay K, Akta? M, Dumanli N. PCR-RFLP analysis of the Tams1 gene of Theileria annulata. Turkiye Parazitology Derg, 2007, 31(3): 173-175.

[14]罗金, 刘光远, 田占成, 谢俊仁, 张萍. 基于18Sr RNA基因序列的我国马梨形虫分类学地位分析. 动物分类学报, 2011, 36(1): 99-103.

Luo J, Liu G Y, Tian Z C, Xie J R, Zhang P. Taxonomic status     of equine piroplasmid assays based on 18S rRNA gene sequencing in China. Acta Zootaxonomica Sinica, 2011, 36(1): 99-103. (in Chinese)

[15]罗金, 刘光远, 田占成, 谢俊仁, 沈辉, 田美媛. 马泰勒虫EMA1基因的克隆及生物学特性分析. 中国兽医科学, 2012, 42(4): 352-356.

Luo J, Liu G Y, Tian Z C, Xie J R, Shen H, Tian M Y. Cloning and phylogenetic analysis of EMA1 gene of Theileria equi. Chinese Veterinary Science, 2012, 42(4):352-356. (in Chinese)

[16]罗金, 刘光远, 田占成, 谢俊仁, 张萍, 沈辉, 党根生. 基于Hsp70 基因的马梨形虫分类学定位分析. 中国农业科学, 2011, 44(17): 3694-3700.

Luo J, Liu G Y, Tian Z C, Xie J R, Zhang P, Shen H, Dang G S. Taxonomic status assays of Theileria equi based on Hsp70   gene. Scientia Agricultura Sinica, 2011, 44(17):3694-3700. (in Chinese)

[17]Bhoora R, Quan M, Matjila P T, Zweygarth E, Guthrie A J, Collins N E. Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGBTM assay for the detection of T. equi. Veterinary Parasitology, 2010, 172: 33-45.

[18]Shiels B R, d'Oliveira C, McKellar S, Ben-Miled L, Kawazu S, Hide G. Selection of diversity at putative glycosylation sites in the immunodominant merozoite piroplasm surface antigen of Theileria parasites. Molecular and Biochemical Parasitology, 1995, 72: 149-162.

[19]M J Gubbels, F Katzer, Hide G, Jongejan F, Shiels B R. Generation of a Bahrain pattern of diversity in the major merozoite-piroplasm surface antigen of Theileria annulata. Molecular and Biochemical Parasitology, 2000, 110: 23-32.

[20]Shiels BR, d’Oliveira C, McKellar S, Ben-Miled L, Kawazu S, Hide G. Selection of diversity at putative glycosylation sites in the immunodominant merozoite: piroplasm surface antigen of Theileria parasites. Molecular and Biochemical Parasitology 1995, 72: 149-162.

[21]Katzer F, McKellar S, Ben Miled L, d’Oliveira C, Shiels B. Selection for antigenic diversity of Tams1; the major merozoite antigen of Theileria annulata. Annals of the New York Academy of Sciences，1998, 849: 96-108.

Related Articles 15

[1]	XU JinQing, BIAN HaiYan, CHEN TongRui, WANG Lei, WANG HanDong, YOU En, DENG Chao, TANG YouLin, SHEN YuHu. Comparison of the Genome Sequence Polymorphisms Between the Main Naked Barley Varieties Kunlun 14 and Kunlun 15 in Qinghai Province [J]. Scientia Agricultura Sinica, 2024, 57(21): 4192-4204.
[2]	LIN Ping, WANG KaiLiang, YAO XiaoHua, REN HuaDong. Development of DNA Molecular ID in Camellia oleifera Germplasm Based on Transcriptome-Wide SNPs [J]. Scientia Agricultura Sinica, 2023, 56(2): 217-235.
[3]	XiaoChuan LI,ChaoHai WANG,Ping ZHOU,Wei MA,Rui WU,ZhiHao SONG,Yan MEI. Deciphering of the Genetic Diversity After Field Late Blight Resistance Evaluation of Potato Breeds [J]. Scientia Agricultura Sinica, 2022, 55(18): 3484-3500.
[4]	LIANG Peng,ZHANG TianWen,MENG Ke,SHAO ShunCheng,ZOU ShiFan,RONG Xuan,QIANG Hao,FENG DengZhen. Association Analysis of the ADIPOQ Variation with Sheep Growth Traits [J]. Scientia Agricultura Sinica, 2022, 55(11): 2239-2256.
[5]	XU ZhiYing,WANG BaiCui,MA XiaoLan,JIA ZiMiao,YE XingGuo,LIN ZhiShan,HU HanQiao. Polymorphism Analysis Among Chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and Wheat 6A and 6D Based on Wheat SNP Chip [J]. Scientia Agricultura Sinica, 2021, 54(8): 1579-1589.
[6]	ZHANG PengFei,SHI LiangYu,LIU JiaXin,LI Yang,WU ChengBin,WANG LiXian,ZHAO FuPing. Advance in Genome-Wide Scan of Runs of Homozygosity in Domestic Animals [J]. Scientia Agricultura Sinica, 2021, 54(24): 5316-5326.
[7]	Yun PENG,TianGang LEI,XiuPing ZOU,JingYun ZHANG,QingWen ZHANG,JiaHuan YAO,YongRui HE,Qiang LI,ShanChun CHEN. Verification of SNPs Associated with Citrus Bacterial Canker Resistance and Induced Expression of SNP-Related Calcium-Dependent Protein Kinase Gene [J]. Scientia Agricultura Sinica, 2020, 53(9): 1820-1829.
[8]	ZHANG QingAn,CHEN BoYu. Research Progress of Four Sulfur Compounds Related to Red Wine Flavor [J]. Scientia Agricultura Sinica, 2020, 53(5): 1029-1045.
[9]	XU Yunbi,YANG QuanNü,ZHENG HongJian,XU YanFen,SANG ZhiQin,GUO ZiFeng,PENG Hai,ZHANG Cong,LAN HaoFa,WANG YunBo,WU KunSheng,TAO JiaJun,ZHANG JiaNan. Genotyping by Target Sequencing (GBTS) and Its Applications [J]. Scientia Agricultura Sinica, 2020, 53(15): 2983-3004.
[10]	CHEN XiaoHong,HE JieLi,SHI TianTian,SHAO HuanHuan,WANG HaiGang,CHEN Ling,GAO ZhiJun,WANG RuiYun,QIAO ZhiJun. Developing SSR Markers of Proso Millet Based on Transcriptome Sequencing [J]. Scientia Agricultura Sinica, 2020, 53(10): 1940-1949.
[11]	XU YueQing,WANG DanDan,ZHAO XinNan,YU Li,WANG XiuPin,ZHANG LiangXiao,ZHANG Qi,ZHANG Wen,LI PeiWu. Advances in Research on Physiological Functions of Vitamin K₁and Its Detection Methods in Agricultural Products [J]. Scientia Agricultura Sinica, 2019, 52(18): 3207-3217.
[12]	CAO XueTao, PEI ShengWei, ZHANG Jin, LI FaDi, LI Gang, LI WanHong, YUE XiangPeng. Screening of Y Chromosome Specific Primers and Y-SNPs in Sheep [J]. Scientia Agricultura Sinica, 2018, 51(15): 2990-2999.
[13]	GAO XiaoLi, HU Jiang, GUO ShuZhen, SHI BinGang, XIE JianPeng, LUO YuZhu, WANG JiQing, MU YongJuan. Polymorphisms of DGAT1 Gene and Their Association with Milk Quality Traits in Yak [J]. Scientia Agricultura Sinica, 2017, 50(16): 3215-3225.
[14]	LIU LiNa, YANG Jing, XU LiuYan, LI ChengYun. Genetic Diversity Analysis of Pi-ta Gene 3′-UTR in Rice Landraces [J]. Scientia Agricultura Sinica, 2017, 50(15): 2851-2860.
[15]	MA Xiao-meng, XUAN Jun-li,WANG Hui-hua,YUAN Ze-hu, WU Ming-ming, ZHU Cai-ye, LIU Rui-zao, WEI Cai-hong, ZHAO Fu-ping, DU Li-xin, ZHANG Li. Association of the RIPK2 Gene Genetic Variation with Ujumqin Sheep Growth Traits [J]. Scientia Agricultura Sinica, 2016, 49(7): 1391-1407.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

Polymorphism Assays of Amino Acid and Establishment of a Two-Temperature PCR for Theileria Annulata Based on Tams1 Gene

PDF