TABLE 1.

Overview of the methods and primers used in the study scheme for molecular detection of T. gondii

Center	DNA extraction method (reference)	DNA target	Primer(s) (reference[s])	PCR technologya	Amplicon detectionb	Inhibition controlc
A	Tween-Noninet-NaOH method (19)	B1 gene	(6)	cnPCR	Gel electrophoresis + ethidium bromide staining	T. gondii DNA internal control
		rep529	H1-H2 (unpublished)	cnPCR	Gel electrophoresis + ethidium bromide staining	T. gondii DNA internal control
B	Qiagen QIAamp DNA mini kit	B1 gene	(29)	cnPCR	ELISA	Plasmidic internal control
		rep529	(33, 38)	rtPCR	TaqMan MGB and LNAe	Plasmidic internal control
C	Roche HighPure PCR template kit	B1 gene	(13)	rtPCR	FRET	β-Globin gene
		rep529	(8)	rtPCR	FRET	β-Globin gene
D	Qiagen QIAamp DNA mini kit	B1 gene	Primers 1-4 (7)	qrtPCR	Sybr Green	Plasmidic internal control
		rep529	(20)	rtPCR	Sybr Green	Plasmidic internal controld
E	Qiagen QIAamp DNA mini kit	B1 gene	(26)	rtPCR	FRET	Plasmidic competitive internal control (5)
F	Qiagen QIAamp DNA blood mini kit	B1 gene	(15)	rtPCR	TaqMan	T. gondii DNA internal control
		rep529	(15)	rtPCR	TaqMan	T. gondii DNA internal control
G	Qiagen QIAamp DNA mini kit	B1 gene	(13)	rtPCR	FRET	β-Globin gene
		rep529	(27)	rtPCR	FRET	β-Globin gene
H	Qiagen QIAamp DNA blood mini kit	rep529	(27)	cnPCR	Gel electrophoresis + ethidium bromide staining	T. gondii DNA internal control
		rep529	(16)	rtPCR	FRET	T. gondii DNA internal control

^acnPCR, conventional PCR; rtPCR: real-time PCR.

^bGel electrophoresis + ethidium bromide staining, direct visualization of DNA by ethidium bromide staining after agarose gel electrophoresis; ELISA, PCR-ELISA based upon the PCR-ELISA DIG labeling and PCR-ELISA DIG detection kits (Roche Diagnostics); TaqMan MGB and LNA, hydrolysis DNA probes (TaqMan technology; Applied Biosystems, Courtaboeuf, France); FRET, FRET hybridization DNA probes; Sybr green, Sybr green-based real-time PCR.

^cThe absence of reaction inhibition was verified by amplifying a positive internal control concurrently and in the same reaction tube as the test DNA after the addition of a control sequence of target DNA; this control DNA may be either highly diluted T. gondii genomic DNA (equivalent to 1 or 0.5 tachyzoite genome), an artificial plasmidic DNA construct containing the primer sequences (amplified by the test primers), or a defined sequence of DNA amplified by a second primer pair, e.g., β-globin or albumin amplified under stringent conditions (to increase the PCR sensitivity to the presence of inhibitors in the sample).

^dLaboratory D also systematically performed one PCR with the matrix DNA diluted.

^eThe two types of probes gave similar results.