Table 3.

The chessboard procedure used to determine how well a multiplex test performs on samples containing >1 target.

Pathogen 1 dilution series	Pathogen 2 dilution series
	(a) 10−1	(b) 10−2	(c) 10−3	(d) 10−4	(e) 10−5	(f) 10−6	(g) 10−7
(A) 10−1	Aa	Ab	Ac	Ad	Ae	Af	Ag
(B) 10−2	Ba	Bb	Bc	Bd	Be	Bf	Bg
(C) 10−3	Ca	Cb	Cc	Cd	Ce	Cf	Cg
(D) 10−4	Da	Db	Dc	Dd	De	Df	Dg
(E) 10−5	Ea	Eb	Ec	Ed	Ee	Ef	Eg
(F) 10−6	Fa	Fb	Fc	Fd	Fe	Ff	Fg
(G) 10−7	Ga	Gb	Gc	Gd	Ge	Gf	Gg
(H) 10−8	Ha	Hb	Hc	Hd	He	Hf	Hg

A dilution series of the extracted nucleic acid from pathogen 1 and pathogen 2 is made. Using a microtitre plate a volume of each dilution of pathogen 1 is added to each column so that, for example, dilution 1 of pathogen 1 (labelled here as (A)) is present in the first well of each column. Subsequently an equal volume of each dilution of pathogen 2 is added to each row so that, for example, dilution 1 of pathogen 2 (labelled here as (a)) is present in the first column only. The wells will now contain different concentrations of pathogens 1 and 2 and can be used to assess the multiplex PCR.