弓形虫ITS-1序列PCR诊断方法的建立

目的本文旨在为弓形虫感染的临床病例检测建立特异性PCR诊断方法。方法本研究以弓形虫ITS-1序列为种特异性遗传标记,采用有限稀释法稀释速殖子DNA,经PCR扩增,检测该方法的敏感性;用同一引物扩增鸡柔嫩艾美耳球虫原虫和弓形虫,检测PCR方法的特异性。人工感染小鼠和家兔,用组织触片法和PCR方法检测感染动物的组织,并检测10头临床疑似病猪的肺脏、肺门淋巴结、脾脏、肾脏和肝脏等组织,比较两者的敏感性。结果该PCR方法最低可以检测1pg弓形虫速殖子DNA(相当于10个速殖子的DNA);鸡柔嫩艾美耳球虫原虫未扩增出特异条带,仅弓形虫出现特异条带(300bp)。组织触片和PCR方法对人工感染小鼠组织DNA的检出率均为87.5%(7/8),对人工感染家兔的检出率分别为50%(3/6)和66.7%(4/6),对临床疑似弓形虫病猪检测的阳性率均为20%,两种方法的检查结果基本一致。结论本试验结果表明,PCR技术可以作为弓形虫感染动物的诊断与检测方法。

Establishment of uPCR assay for diagnosis of Toxoplasma gondii infection

To establish a special PCR assay to detect the presence of Toxoplasma gondii,the 18S-5.8S rRNA internal transcribed spacer-1(ITS-1)region was used as the special genetic marker in this test.The tachyzoite DNA was diluted by limiting dilution assay and after PCR amplification it was used to detect the sensitivity of PCR assay.In order to detect its specificity,PCR amplification using the same primer pairs was performed with DNA from geographic Eimeria tenella in chicken and T.dondii.The tissue smear method and PCR assay were simultaneously employed to detect the presence of T.gondii both in tissues of the experimentally infected mice and rabbits and in the clinical samples including lungs,brocho-pulmoanry lymph nodes,spleen,kidney and liver of 10 pigs with suspected toxoplasmosis,thus to compare its sensitivity of testing with these two method of assay.It was found that the lowest value to detect the T.gondii DNA was 1 pg of tachyzoite DNA(equivalent to amount of DNA from 10 tachyzoites),in which the specific band of 300 bp targeting the ITS-1 region appeared only on DNA from T gondii and no specific band could be detected with DNA from E,tenella in chicken.The detection rate of T.gondii by using tissue smear method and PCR assay were both 87 5%(7/8),but those for tissues from experimentally infected mice and rabbits were 50%(3/6)and 66.7%(4/6)respectively.However,the positive rate of detection for T.gondii in 10 pigs with suspected toxoplasmosis by using tissue smear method and PCR assay both were 20%.It is evident that the results by using these two kinds of methods to detect the presence of T.gondii are considerably consistent,suggesting that the PCR assay can be applied for the diagnosis of T.gondii infection in animals.