基于529bp高度重复序列的弓形虫PCR诊断方法的建立

目的建立基于529bp高度重复序列的弓形虫PCR诊断方法。方法以弓形虫基因组中529bp的高度重复序列为诊断靶基因设计引物,并以其为模板经PCR扩增该基因,与pMD18-T载体连接,构建pMD18/Tox529bp重组质粒,测序鉴定正确后,经稀释标定作为标准品。优化PCR反应体系及条件,并验证该方法的灵敏度及特异性。结果所构建的pMD18/Tox529bp重组质粒经测序正确,建立的PCR方法最低可检出10个拷贝的目的基因,除对弓形虫基因组DNA扩增出特异性条带外,用该方法对健康志愿者全血、正常小鼠全血、间日疟原虫、恶性疟原虫及结核杆菌基因组DNA均未扩增出特异性条带。结论已建立了一种具有较高灵敏度和特异性的弓形虫PCR诊断方法 ,有望应用于人和动物弓形虫感染的筛查、临床诊断及流行病学调查。

Development of A PCR Assay for Diagnosis of Toxoplasma gondii Infection Based on Repetitive 529 bp Fragment

Objective To develop a PCR assay for diagnosis of Toxoplasma gondii infection based on repetitive 529 bp fragment.Methods Primers were designed and synthesized according to the repetitive 529 bp fragment of genome of Toxoplasma gondii,based on which the fragment was amplified by PCR and inserted into vector pMD18-T.The constructed recombinant plasmid pMD18 /Tox529 bp was identified by sequencing,then diluted,calibrated and used as standard.The reaction system and condition for PCR were optimized,and the sensitivity and specificity of the developed PCR assay were verified.Results Sequencing proved that recombinant plasmid pMD18 /Tox529 bp was constructed correctly.The minimum detection limit of the developed PCR assay was 10 copies of target gene.Specific DNA bands were amplified by the developed PCR assay from the genome of Toxoplasma gondii.However,no specific DNA bands were amplified from the genomes of human,mouse,Plasmodium vivax,Plasmodium faciparum or Mycobacterium tuberculosis.Conclusion A highly sensitive and specific PCR assay for diagnosis of Toxoplasma gondii was developed,which might be used for the screening,clinical diagnosis and epidemiological investigation of Toxoplasma gondii infections in humans and animals.