Table 1.

Theoretical detection levels of the various LCMV detection methods in organs of immune mice

Detection limit
Focus-forming assay	Nested NPRT-PCR	Transfer in AG 129 mice
40–400 pfu*	100–200 copies†	2–4 infective units‡

The indicated detection limits for three different LCMV detection methods can be calculated from the following dilution factors and optimal sample volumes used for each technique: 

^*As only a quarter of spleen (1/4) was harvested for the focus forming assay, yielding 2 ml of tissue homogenate from which only 200 μl (1/10) were used in the assay, the theoretical detection level of the focus-forming assay is 40 pfu if one plaque is detected in the first dilution step and 400 pfu if the cell layer in the first dilution well is destroyed by the toxic effects of the homogenate. 

^† Only 8 μl of the total 40-μl RNA solution extracted from a quarter spleen were used for the reverse transcription reaction (1/5), and 2 μl of the resulting total of 16 μl cDNA (1/8) were used in the PCR reaction. The theoretical detection limit if one copy gives a positive signal would therefore be 160 copies per spleen. 

^‡ We transferred half or a quarter of the whole amount of tissue homogenate in AG 129 mice. If one infective unit of LCMV leads to viremia in these mice, a minimum of 2–4 units would be expected in the whole organ.