弓形虫特异DNA片段顺序分析及体外基因扩增

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。

Sequencing of Specific DNA Fragment from the Toxoplasma Gondii and in Vitro Gene Amplification

We have screened out a specific Toxoplasma gondii DNA fragment from a genomic DNA library of T.gondii (ZS2 strain).The partial sequences of the cloned segment were analysed.A specific primer pair of the oligonucleotides for the T.gondii DNA sequences has been designed and synthesized in our laboratory.Polyme- rase chain reaction for in vitro enzymatic amplifying the specific DNA sequence for T.gondii was established.A specific amplified band was shown in the PCR products from DNAs from four different strains of T.gondii and peripheral white blood cells and thymus of three baby pigs artificially infected with T.gondii, but was not shown in DNAs from controls, ie., normal human and baby pig peripheral white blood cells, spleen of normal mouse, plasmodium falciparum, Pneumocystis carinii, Entameba histolytica and human cytomegalovirus.The amplified products were further analysed by southern blot and restriction pattern and shown to be T.gondii origin.As little as one parasite of T.gondii or one pg of purified DNA from T.gondii can be detected by the PCR method.The analysed DNA sequences of the cloned fragment and the designed primers are different from all the known DNA sequence in the gene bank.This is a simple, sensitive and rapid method for detecting T.gondii.