Basic information

Latin name Ambystoma cingulatum & Ambystoma bishopi
Common name Frosted and Reticulated Flatwoods Salamanders
Broad taxonomic group amphibian
eDNA source water
assay type qPCR
gene/region mitochondrial d-loop
5′ reporter dye FAM
TaqMan MGBNFQ (minor groove binder non fluorescent quencher) yes
PCR chemisty Qiagen QuantiTect Multiplex PCR NoROX Kit (Qiagen, Inc.)
latest publication year 2015
Number of citations 1
Citation 177

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 1
detection obtained from environmental samples detection from an environmental sample (artificial or natural habitat) 1
limit of detection (LOD) established LOD determined 0
extensive field testing of environmental samples multiple locations or multiple samples 1
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 1
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 0
geographic scope 0
number of individuals the tissue was extracted from 1
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 0
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) 1
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 1
optimization of PCR conditions 0
how many per species 1
geographic origin 0
tissue source (fresh or old) 0
non-target amplification checked via sequencing 0
DNA concentration 0
basic geographic scope 1
unidirectional lab flow 1
extensive geographic testing 0
method of extraction 1
modifications from established protocol 0
lysate volume 0
extraction negative control 1
elution volume 0
verification of DNA extraction 0
volume weight of environmental sample 1
filter type 1
pore size 1
surface area diameter 0
pressure used for filtration 0
filter preservation 1
precipitation chemicals added NA
centrifugation force and time for precipitation NA
temperature of precipitation NA
storage between collection and processing 1
“field” blanks 1
criteria to determine detection 1
sample type (e.g., water sediment) 1
artificial habitat 0
natural habitat 1
target DNA spiked into sample type 0
environmental variables recorded 1
sequence validation of positive field samples 0
determined limit of detection 0
method of establishment NA
multiple replicates of standard curve NA
multiple locations 1
site where species is not there 0
site where species is there 1
site where species was there historically 0
multiple samples at each site 1
environmental gradients (e.g., abiotic biotic conditions) 1
different known target species densities 0
testing against data from traditional methods 1
inhibition testing 1
geographic region tested 0
source of species occurrence information 0
multiple samples per species 1
geographic origin of tissue samples 0
tissue source 0
non-target amplification checked via sequencing 0
DNA concentration 0
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 1
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