TABLE 3.

Standardization of limiting-dilution nested nef-p15 multiplex PCR^a

Baby	Tissue	Time point (wk)	DNA per reaction (ng)b	No. of replicates	No. positive	Total ng of DNA tested	Estimated copy no./μg DNAc	Estimated copy no./million cellsc,d	Correlation Coefficiente
B1	Inguinal lymph node	24	20.6000	6	6	123.60000	150,403	992,662	0.97
			4.12000	6	6	24.72000
			0.82400	6	6	4.90000
			0.16400	6	6	0.99000
			0.03300	6	6	0.20000
			0.00700	6	6	0.04000
			0.00130	6	3	0.00800
			0.00030	6	2	0.00200
						0.00005				6	0	0.00030
						0.00001				6	0	0.00006
B1	Thymus	24	500.000b	6	6	3,000.000	3,119	20,586	0.75
			100.000	6	6	600.000
			20.000	6	6	120.000
			4.000	6	6	24.000
			0.800	6	5	4.800
			0.160	6	4	0.960
			0.032	6	0	0.192

^aLimit of detection of nested nef-p15 PCR assay: 2 to 5 copies/μg DNA. The limit of detection for the nested multiplex PCR was estimated by performing multiple independent limiting-dilution nested nef-p15 PCRs on a standard panel of templates that contained 1, 10, 100, and 1,000 copies/PCR.

^bThe amount of DNA used in the PCR standardization assays was based on the virus load estimated using real-time DNA PCR. The starting amount was 2,000 copies/μg DNA, with fivefold dilutions to less than 1 theoretical copy/μg DNA.

^cThe copy number per microgram of DNA was estimated using Quality Software (24).

^dThe estimated copy number per microgram DNA was extrapolated to the copy number per million cells based on the amount of DNA per cell (6.6 pg DNA per cell).

^eCorrelation coefficient between viral copies per microgram DNA estimated from Quality Software and the real-time DNA virus load.