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从弓形虫(ZS_2株)基因组文库中筛选出了一个弓形虫特异DNA片段的克隆,对克隆的片段进行了部分顺序分析。根据所得DNA顺序,自行设计并合成特异的寡核苷酸引物对,建立了体外扩增弓形虫特异DNA顺序的聚合酶链反应(PCR)方法。4种不同来源的弓形虫株DNA、人工感染弓形虫的三头幼猪白细胞和胸腺DNA经过PCR扩增,均出现特异的扩增条带;而正常人、正常幼猪外围血白细胞、正常小鼠脾脏、恶性疟原虫、卡氏肺孢子虫、溶组织内阿米巴和人巨细胞病毒DNA均不出现特异的扩增条带。对扩增产物进行了Southern印迹和限制性内切酶图谱分析,证明该PCR产物是弓形虫DNA特异的顺序。该方法可测出少至1pg的弓形虫DNA或1个弓形虫体的裂解液。本文分析的DNA顺序和设计合成的引物顺序数据,经电脑DNA数据库检索,证明无相同的顺序。本方法并具有简便、快速等优点,便于推广应用。
A Toxoplasma gondii-specific DNA fragment was screened from the genomic library of Toxoplasma gondii (ZS 2 strain), and the cloned fragments were partially sequenced. According to the obtained DNA sequences, specific oligonucleotide primer pairs were designed and synthesized, and a polymerase chain reaction (PCR) method for amplifying Toxoplasma gondii specific DNA sequences in vitro was established. Toxoplasma gondii DNA from four different sources, three piglets infected with Toxoplasma gondii were amplified by PCR, and the specific amplified bands appeared. However, the peripheral blood leukocytes in normal and normal piglets were normal Spleen, Plasmodium falciparum, Pneumocystis carinii, Entamoeba histolytica and human cytomegalovirus DNA showed no specific amplification bands. Southern blotting and restriction endonuclease mapping of the amplified product proved that the PCR product was DNA-specific to Toxoplasma gondii. This method can detect as little as 1 pg of Toxoplasma gondii DNA or one Toxoplasma lysate. The DNA sequence analyzed in this paper and the sequence data of the designed and synthesized primers were searched by the computer DNA database to prove that there is no identical sequence. The method has the advantages of simplicity, rapidness, etc., and is convenient for popularization and application.