TABLE 2.

Technical characteristics of var group primers for SYBR green real-time PCR^a

Characteristic	Value or description for indicated primer pair					Value or description for indicated primer pair
	A1	A2	A3	B1	B2	C1	C2	BC1	BC2	pvar1utr	pvar2utr	pvar3
Target region	Grp A DBL1α	Group A exon 2	Group A exon 2	upsB	upsB	upsC subtype	upsC subtype	Group B and C exon 2	Group B and C exon 2	var1 5′ UTR	var2 5′ UTR	var3 coding
Targeted genes in 3D7 predicted by alignments	PFD1235w, MAL7P1.1, PF08_0141, PF11_0008, PFD0020c, PF11_0521, PF13_0003, PFA0015c, MAL6P1.314, PFI1820w	PFD1235w, MAL7P1.1, PF08_0141, PF11_0008, PFD0020c, PF11_0521, PF13_0003, PFA0015c, MAL6P1.314, PFI1820w	PF11_0521, PFD1235w, MAL7P1.1, PF13_0003, PFD0020c, PF11_0008	MAL6P1.1, PF07_0139, PF08_0142, PF10_0001, PF10_0406, PF11_0007, PF13_0001, PF13_0364, PFA0005w, PFA0765c, PFB0010w, PFB1055c, PFC0005w, PFC1120c, PFD0005w, PFD1245c, PFE0005w, PFI0005w, PFI1830c, PFL0005w, PFL0935c, PFL2665c	MAL6P1.1, PF07_0139, PF08_0142, PF10_0001, PF10_0406, PF11_0007, PF13_0001, PF13_0364, PFA0005w, PFA0765c, PFB0010w, PFB1055c, PFC0005w, PFC1120c, PFD0005w, PFD1245c, PFE0005w, PFI0005w, PFI1830c, PFL0005w, PFL0935c, PFL2665c	PFD0625c, PFL1960w, PF07_0048, PFD0630c, PF07_0049, PF07_0051, PFD0615c, PFD1015c	PFD0615c, PF07_0051, PFD0995c, PFD1000c, PF07_0049, PFD1015c, MAL6P1.252	PF08_0142, MAL6P1.1, PFA0765c, PFC1120c, PFC0005w, PFE0005w, PF10_0406, PFB1055c, MAL7P1.56, PF08_0140, PF13_0364, PF13_0001, PFL2665c, PFI0005w, PFB0010w, PF11_0007, PF07_0139, PFD1005c, PFD1000c, PF08_0107, PFD0005w, PFL0005w, PFD1015c, PFI1830c, PF07_0050, PF10_0001	MAL7P1.50, PF07_0048, PF08_0103, PF08_0106, PFA0005w, PFD1245c, PFL0935c, PFL1950w	PFE1640w	PFL0030c	PFA0015c, MAL6P1.314, PFI1820w
Coverage in 3D7 (group)	All A genes, including var3 types	All A genes, including var3 types	All A genes, excluding var3 types	All B genes	All B genes	8/13 C	7/13 C	17/22 B, 4/13 C, 1/4 BA, 2/9 BC	4/9 BC, 3/22 B, 1/13 C	var1	var2	var3
Fragment Tm (°C) in 3D7	77.2	73.6	72.1	75.4	75.6	74.1	73.5	75.9	75.5	69.6	74	75.5
Fragment length (bp)	110-120	100	160	260	190	106	120	110	170	87	184	155
Fragment Tm (°C)b in field isolates (SD) [no. of PCR-negative genomes/total]	77.2 (0.4) [0/20]	73.7 (0.2) [0/20]	72.2 (0.4) [0/20]	75.1 (0.4) [0/20]	75.3 (0.4) [0/20]	74.2 (0.4) [0/20]	73.5 (0.3) [0/20]	75.9 (0.3) [0/20]	75.6 (0.3) [0/20]	69.6 (0.2) [0/20]	74.3 (0.4) [0/20]	75.6 (0.2) [0/20]
[−log(no. of targeted genes)/log(2)]	−3.3	−3.3	−2.6	−4.5	−4.5	−3	−2.8	−4.7	−3	0	0	−1.6
)	−2.0	−2.5	−1.9	−3.7	−3.9	−2.2	−0.7	−4.5	−1.67	2.8	−0.8	−1.8
[μ( − ) (95% CI)]c	−1.2 (0.4)	−1.9 (0.3)	−1.9 (0.3)	−4 (0.2)	−3.6 (0.2)	0.3 (0.8)	0.9 (0.5)	−4.55 (0.2)	0.7 (0.3)	2.6 (0.2)	0.3 (0.3)	ND

^aThe following 3D7 var genes were not targeted by any primer pair: PFD0635c, PF07_0050, PFL1955w, MAL7P1.55, MAL6P1.4, MAL6P1.316, and PFL0020w (three BA and four BC genes).

^bMean Tm based on real-time PCR on 20 gDNA and 108 cDNA field isolates.

^c95% confidence intervals based on real-time PCR on gDNAs from 20 field isolates. The design and validation of primers and probes for the MGB real-time PCR assay were described by Kaestli et al. (16).