Basic information

Latin name Anaxyrus fowleri
Common name Fowler’s Toad
Broad taxonomic group amphibian
eDNA source tissue tests only
assay type qPCR
gene/region Cytochrome c oxidase subunit I
5′ reporter dye VIC
PCR chemisty TaqMan Environmental 2.0 Master Mix (Life Technologies)
latest publication year 2018
Number of citations 1
Citation 16

Validation score and minimum criteria

Validation level


Variable blocks Minimum criteria Score
in silico analysis target species 1
target tissue testing target tissue 1
target tissue PCR primer (and probe) sequences reported 1
comprehensive reporting of PCR conditions DNA extract volume in PCR 1
in vitro testing on closely related non-target species any in vitro testing 1
extraction method performed on environmental DNA samples method of extraction 1
concentration of eDNA from environmental sample filter type or precipitation chemicals 0
detection obtained from environmental samples detection from an environmental sample (artificial or natural habitat) 0
limit of detection (LOD) established LOD determined 1
extensive field testing of environmental samples multiple locations or multiple samples 0
in vitro testing on co-occurring non-target species any advanced in vitro testing 1
comprehensive specificity testing non-co-occurring / closely related species checked from in silico 0
detection probability estimation from statistical modelling any effort made towards detection probability estimation 0
understanding ecological and physical factors influencing eDNA in the environment any factor influencing eDNA in the environment tested 0

Detailed validation scores

database reported 1
target species 1
software name 1
software parameters 0
how many mismatches etc. 1
where are mismatches located 3′ 5′ 0
list potential non target organisms that may cause problems 0
closely related 1
co-occurring 1
target tissue 1
tissue source 1
geographic scope 1
number of individuals the tissue was extracted from 0
tested on more than one haplotype 0
DNA concentration of the target extracts 0
primer (and probe) sequences reported 1
thermocycling conditions 1
check of amplification success 1
amplification confirmed by sequencing 1
gene / region reported 1
probe (if applicable; 5′-3′) 1
5′ reporter dye (e.g. FAM, VIC, TET, NED) 1
internal quencher (in the middle of the probe sequence; e.g. ZEN) NA
3′ quencher (ZEN, BHQ1, BHQ2, IBFQ, TAMARA) 0
TaqMan MGBNFQ (minor groove binder non fluorescent quencher) (yes/no) 0
volume 1
primer concentration 1
use of commercial Master Mix 1
use of custom Master Mix 0
MgCl2 concentration NA
dNTP concentration NA
buffer type and concentration NA
enzyme type and concentration 0
enhancer chemicals (additives TMAC, BSA, DIMSO….) 0
DNA extract volume in PCR 1
cycler model 1
annealing time 1
annealing temp 1
cycles 1
technical replication 0
optimization of PCR conditions 1
how many per species 0
geographic origin 1
tissue source (fresh or old) 1
non-target amplification checked via sequencing 0
DNA concentration 1
basic geographic scope 1
unidirectional lab flow 0
extensive geographic testing 0
method of extraction 1
modifications from established protocol 0
lysate volume 0
extraction negative control 0
elution volume 1
verification of DNA extraction 1
volume weight of environmental sample NA
filter type NA
pore size NA
surface area diameter NA
pressure used for filtration NA
filter preservation NA
precipitation chemicals added NA
centrifugation force and time for precipitation NA
temperature of precipitation NA
storage between collection and processing NA
“field” blanks NA
criteria to determine detection NA
sample type (e.g., water sediment) NA
artificial habitat NA
natural habitat NA
target DNA spiked into sample type NA
environmental variables recorded NA
sequence validation of positive field samples NA
determined limit of detection 1
method of establishment 1
multiple replicates of standard curve 0
multiple locations NA
site where species is not there NA
site where species is there NA
site where species was there historically NA
multiple samples at each site NA
environmental gradients (e.g., abiotic biotic conditions) NA
different known target species densities NA
testing against data from traditional methods NA
inhibition testing NA
geographic region tested 1
source of species occurrence information 0
multiple samples per species 0
geographic origin of tissue samples 0
tissue source 0
non-target amplification checked via sequencing 0
DNA concentration 1
non co-occurring closely related species checked from in silico 0
justify non-verified results from in silico (geographic realistic) 0
non-target amplification checked via sequencing 0
sampling through time 0
sampling spatially 0
statistical modelling 0
occupancy modelling 0
parameters and variables of model reported 0
know origin (e.g., shedding rate) 0
state 0
degradation rate 0
transport 0