Primer design and detection of diluted DNA samples from an infected chicken.

A and B, Aligned primer sequences based on genes encoding mitochondrial cytochrome b (cytb, A) and cytochrome oxidase subunit III (coxIII, B) from L. sabrazesi (NCBI accession No. AB299369.1), Leucocytozoon caulleryi (Accession No. AB302215.1), Haemoproteus columbae (NCBI accession No. FJ168562.1), Plasmodium gallinaceum (Accession No. AB250690.1), and chicken (Gallus gallus domesticus, Accession No. KM096864.1). C, Amplifications of diluted DNA from an infected chicken with known gametocytemia. DNA sample from infected chicken (#2HC with parasitemia of 0.02%) blood obtained from Haicang, Fujian province, was diluted in water at ratios of 1:10; 1:50; 1:200; 1:1,000; 1:5,000; 1:20,000; 1:100,000; 1:500,000; 1:2×10⁶; and 1:1×10⁷ and was amplified using Ls-coxIIIF2/R2 and Ls-cytbF1/R1 primers, respectively. PCR products (4 μl each) were separated on a 2% agarose gel. A DNA band could be easily detected at 1:1,000 dilution using Ls-coxIIIF2/R2, and a band at 1:5,000 could be seen using the Ls-cytbF1/R1 primers. “+” indicates un-diluted DNA control. The figure is representative of two replicates with the same results.