Figueroa J V, Chieves L P, Johnson G S, Buening G M
Department of Veterinary Microbiology, University of Missouri-Columbia 65211.
Vet Parasitol. 1993 Oct;50(1-2):69-81. doi: 10.1016/0304-4017(93)90008-b.
A highly sensitive polymerase chain reaction (PCR) based method was developed to detect, in the same blood sample, DNA of hemoparasites frequently found together infecting cattle in tropical and subtropical areas. Bovine blood containing equal parasitemias of Babesia bigemina, B. bovis and Anaplasma marginale infected erythrocytes was mixed to standardize the test. Twenty microliters of 10-fold dilutions from the pooled blood sample were resuspended in PCR mixture buffer containing each of the species-specific sets of primers. Group I primers (BiIA/IB, BoF/R and Am9/10) which specifically bind B. bigemina, B. bovis and A. marginale DNA were used to amplify a fragment of DNA from genomic parasite DNA. Group II nested primers (BiIAN/IBN, BoFN/RN and Am11/12) were used to prepare, via incorporation of digoxigenin-11-dUTP by PCR, nonradioactive probes specific for internal sequences present in DNA amplified with Group I primers. Agarose gel electrophoresis and Southern blot hybridization studies showed that by using Group I primers, DNA fragments of 278 bp, 350 bp and 200 bp were specifically amplified in samples containing B. bigemina, B. bovis and A. marginale DNA, respectively. The analytical sensitivity of the multiple PCR test, as evaluated by nucleic acid hybridization with the nonradioactive probe, was 0.00001%, 0.00001% and 0.0001% infected erythrocytes for B. bigemina, B. bovis and A. marginale, respectively. Blood collected from cattle previously inoculated with B. bovis (4 years), A. marginale (2 years) and B. bigemina (1 year) was demonstrated to be latently infected by using the Multiplex PCR test.
开发了一种基于高灵敏度聚合酶链反应（PCR）的方法，用于在同一血液样本中检测热带和亚热带地区牛群中常见的一起感染的血液寄生虫DNA。将含有等量双芽巴贝斯虫、牛巴贝斯虫和边缘无浆体感染红细胞的牛血混合，以标准化检测。从混合血液样本中取20微升10倍稀释液，重悬于含有每种物种特异性引物组的PCR混合缓冲液中。用于特异性结合双芽巴贝斯虫、牛巴贝斯虫和边缘无浆体DNA的I组引物（BiIA/IB、BoF/R和Am9/10）用于从基因组寄生虫DNA中扩增一段DNA片段。II组巢式引物（BiIAN/IBN、BoFN/RN和Am11/12）用于通过PCR掺入地高辛-11-dUTP来制备对用I组引物扩增的DNA中存在的内部序列具有特异性的非放射性探针。琼脂糖凝胶电泳和Southern印迹杂交研究表明，使用I组引物时，在分别含有双芽巴贝斯虫、牛巴贝斯虫和边缘无浆体DNA的样本中特异性扩增出了278 bp、350 bp和200 bp的DNA片段。通过与非放射性探针进行核酸杂交评估，多重PCR检测对双芽巴贝斯虫、牛巴贝斯虫和边缘无浆体的分析灵敏度分别为0.00001%、0.00001%和0.0001%感染红细胞。使用多重PCR检测证明，从先前接种过牛巴贝斯虫（4年）、边缘无浆体（2年）和双芽巴贝斯虫（1年）的牛采集的血液存在潜伏感染。
