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.

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

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