Table 2.

Nested PCR primers and cycling conditions used to amplify whole mitochondrial genome of haemosporidian parasites

PCR	Forward (F) and reverse (R) PCR primers	Modified after	Fragment size (bp)	Cycle conditions: temperature (°C)/time (s) for denaturation, annealing and extension steps
1 outer	F: AGACGTTCTTAACCCAGCTCACG	346F (Perkins 2008)	1.450	94/30, 58/30, 72/100
	R: CCTTTCCGGCTGTTTCCATCTC
1 inner	F: ATTGGTAAGGTATAGCGTTTACTATCG		1.300	94/30, 57/30, 72/100
	R: GACGAGCGGTGTGTACAAGGC
2 outer	F: CCAACAGAAAAATATTTTAAAGATG		2.050	94/30, 48/30, 72/120
	R: GAACATATCATATTCCAACCATTTA
2 inner	F: ATAAAGAATATTATTTATAAGAACGG		1.900	94/30, 48/30, 72/120
	R: CCAAGGAAATGCATAGGTAA
3 outer	F: TTTATATGTACATTTACTTTTGGWGG		2.020	94/30, 51/30, 72/120
	R: CTTGGAAGATTCGTAATTAGTGG
3 inner	F: GCTTTACATGATACATATTATGTAATTGC	= 3180F (Perkins 2008)	1.900	94/30, 53/30, 72/120
	R: GAATATAGACGGTTTTCTGCG
4 outer	F: TTAGCAAGACATGACAGGG		1.810	94/30, 51/30, 72/100
	R: GGAAAAGGAAAGGTTAACCGC
4 inner	F: GGCAAGTTAAAGAAGTTCTGGTTT		1.750	94/30, 56/30, 72/100
	R: GGGAAGTGTGTTTCCATAGAAACCTTC	626R (Perkins 2008)

All outer reactions included an initial denaturation period of 4 min at 94 °C and 25 cycles. Nested reactions included an initial denaturation period of 4 min at 94 °C and 35 cycles. All reactions included a final extension period of 7 min at 72 °C