A multiplex PCR (mPCR) assay for simultaneous detection and differentiation of four major haemoparasites in crossbred cattle was established using parasite specific genomic DNA and four sets of primer pairs targeting AMA-1, Tams1, MSP5 and VSG genes of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi generating precise amplicons of 448, 156, 382 and 110 bp, respectively. An internal amplification control, 202 bp bovine β-casein gene fragment, was simultaneously amplified with four target genes to avoid false-negative results. The sensitivity of mPCR was 3.44 × 10², 5.9 × 10³, 2.88 × 10² and 3.3 × 10³ copies for B. bigemina, T. annulata, A. marginale and T. evansi, respectively. mPCR of cattle clinical samples (n = 516), suspected for haemoparasites, revealed single haemoparasitic infection in 279 (54.06%) cases, whereas mixed infection was recorded in 54 (10.46%) samples. In clinical samples, coinfection with T. annulata and A. marginale was the most common. The findings of mPCR were consistent with uniplex PCR under field conditions except for subtle variations in A. marginale infection. Overall, the mPCR assay represents an economical, reproducible and robust diagnostic tool for concurrent detection of cattle haemoparasites and large scale epidemiological studies.

多重PCR（多重PCR）测定法同时检测和在杂交牛四大haemoparasites使用寄生虫特异的基因组DNA成立以及四组引物对靶向的分化AMA-1 ，Tams1，MSP5和VSG的基因巴贝斯bigemina，环形泰勒虫，边缘无浆质虫和伊万氏锥虫分别产生448、156、382和110 bp的精确扩增子。内部扩增对照（202 bp牛β-酪蛋白基因片段）与四个靶基因同时扩增，以避免假阴性结果。多重PCR的灵敏度为3.44×10 ²，5.9×10 ³，2.88×10 ²和3.3×10^3个副本B. bigemina，T.菇，A.边缘无和T.伊氏锥虫，分别。怀疑存在血寄生虫的牛临床样本（n = 516）的mPCR显示279例（54.06％）病例中有单一血寄生虫感染，而54例（10.46％）样本中记录到混合感染。在临床样本，共感染与T.菇和A.边缘无是最常见的。在田间条件下，mPCR的发现与单重PCR一致，除了边际曲霉的细微变化感染。总体而言，mPCR分析代表了一种经济，可重复且强大的诊断工具，可同时检测牛血寄生虫和进行大规模的流行病学研究。