Fig. 3
Agarose gel electrophoresis separation of PCR products of the Babesia bigemina PCR detection methods: Two replica comparisons of the relative sensitivity of the nested PCR and semi-nested PCR methods for the detection of B. bigemina DNA using genomic DNA extracted from in vitro cultured parasites from the B. bigemina Puerto Rico strain (A) and from an experimentally infected bovine with the B. bigemina Puerto Rico strain (B). a. The two PCR methods used were based on the amplification of a B. bigemina specific (U) and on the rap-1c (L) genes. The amounts of DNA amplified were 5 ng (lanes 1 and 8); 5×10−1 ng (lanes 2,8); 5×10−2 ng (lanes 3,9); 5×10−3 ng (lanes 4,10); 5×10−4 ng (lanes 5, 11); 5×10−5 ng (lanes 6,12); and 5×10−6 ng (lanes 7,13); lane 14 represents PCR amplifications without adding DNA, and lane 16 represents PCR amplifications using plasmid pCR™ 2.1-TOPO B. bigemina-1 (U) or plasmid pCR™ 2.1-TOPO B. bigemina-rap-1c (L) as positive controls. b. The two PCR methods used were based on the B. bigemina gene (U) and on the rap-1c (L) genes. The amounts of DNA amplified were 5 ng (lanes 1,10); 5x10−1 ng (lanes 2,11); 5×10−2 ng (lanes 3,12); 5×10−3 ng (lanes 4,13); 5×10−4 ng (lanes 5,14); 5×10−5 ng (lanes 6,15); 5×10−6 ng (lanes 7,16); 5×10−7 ng (lanes 8,17); and 5×10−8 ng (lanes 9, 18). Lane 19 represents PCR amplification without adding DNA, and lane 20 represents PCR amplifications using plasmid pCR™ 2.1-TOPO B. bigemina (U) or plasmid pCR™ 2.1-TOPO B. bigemina-rap-1 (L) as positive controls. Molecular size ladder in base pair (bp), 100 bp DNA ladder (lane m). The amplicons of interest are indicated at the left