. 2014 Jan 3;9(1):e84743. doi: 10.1371/journal.pone.0084743

Table 3. Results of the blind testing of single and mixed infections of pospiviroids in plant samples, showing a comparison of three separate methods for setting thresholds for positivity.

	Assays of the Luminex MagPlex-TAG pospiviroid array^a,b,c
Samples	CSVd	CEVd	CLVd	IrVd-1	PCFVd	PSTVd	TASVd	TCDVd	TPMVd	PospUni	PlantIC
CSVd	415**	55	77	264	161	100	623	247	108	1001	10057***
CEVd +TCDVd	172	42541***	186	390	526	171	2038	6236***	380	52636***	25679***
CLVd +TCDVd	158	132	2328**	1013	1081	142	4707*	578**	680	12640***	58345***
IrVd-1+ PSTVd+TCDVd	133	121	187	28821***	427	489**	1783	423**	409	24079***	14937***
PCFVd	88	61	245	562	2425**	150	3311*	172	340	2372**	40987***
PSTVd	193	93	198	287	467	7794***	1625	288	191	16183***	23642***
TASVd	177	123	391	520	476	209	22959***	172	239	21317***	23596***
TCDVd	109	62	216	457	404	97	2736	445**	342	2970**	29558***
TPMVd+PSTVd	149	85	180	409	339	636**	1366	87	7759***	17403***	8101***
Healthy tomato	143	112	243	668	687	155	2965	207	370	845	44083***
Healthy tomato	111	159	349	792	879	76	5250*	206	409	903	61464***
No template control	69	48	37	168	106	65	80	49	67	23	26

^a Mean median fluorescence intensity (MFI) values are presented for each assay of the array.

^b Acronyms refer to assays of the multiplexed array specific for: CSVd - Chrysanthemum stunt viroid; CEVd - Citrus exocortis viroid; CLVd - Columnea latent viroid; IrVd-1 - Iresine viroid 1; PCFVd - Pepper chat fruit viroid; PSTVd - Potato spindle tuber viroid; TASVd - Tomato apical stunt viroid; TCDVd - Tomato chlorotic dwarf viroid; TPMVd - Tomato planta macho viroid; PospUni - pospiviroid universal assay; PlantIC - plant internal control assay.

^c Asterisks indicate values that exceed the threshold for positivity using one or more of the three threshold setting methods compared in this analysis. Method 1 utilizes the novel, non-parametric data-driven threshold setting method described in this study. Method 2 utilizes the arbitrary threshold cut-off set at the value of the mean MFI plus two-fold standard deviation of healthy (uninfected) control samples. Method 3 utilizes the arbitrary threshold cut-off set at the value of the two times the mean MFI of healthy (uninfected) control samples, calculated separately for each assay of the array. Values with three asterisks denote positive samples correctly identified using all threshold-setting methods (Methods 1, 2 and 3). Values with two asterisks denote positive samples correctly identified using Methods 1 and 3 only (and thereby denote false negative samples using Method 2). Values with one asterisk denote false positive samples identified using Method 2.