Standard PCR

Basic information

Latin name Ancylus fluviatilis
Common name
Broad taxonomic group mollusk
eDNA source water
assay type standard PCR
gene/region Cytochrome c oxidase subunit I
latest publication year 2015
Number of citations 2
Citation 162 & 163

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 0
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 0
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 1
how many mismatches etc. 1
where are mismatches located 3′ 5′ 0
list potential non target organisms that may cause problems 1
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 1
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′) NA
5′ reporter dye (e.g. FAM, VIC, TET, NED) NA
internal quencher (in the middle of the probe sequence; e.g. ZEN) NA
3′ quencher (ZEN, BHQ1, BHQ2, IBFQ, TAMARA) NA
TaqMan MGBNFQ (minor groove binder non fluorescent quencher) (yes/no) NA
volume 1
primer concentration 1
use of commercial Master Mix 0
use of custom Master Mix 1
MgCl2 concentration 1
dNTP concentration 1
buffer type and concentration 1
enzyme type and concentration 1
enhancer chemicals (additives TMAC, BSA, DIMSO….) 1
DNA extract volume in PCR 1
cycler model 0
annealing time 1
annealing temp 1
cycles 1
technical replication 0
optimization of PCR conditions 1
how many per species 0
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 1
extraction negative control 1
elution volume 1
verification of DNA extraction 0
volume weight of environmental sample 1
filter type 1
pore size 1
surface area diameter 1
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 0
sequence validation of positive field samples 1
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) 0
different known target species densities 0
testing against data from traditional methods 1
inhibition testing 0
geographic region tested 0
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 NA
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 0
statistical modelling 1
occupancy modelling 0
parameters and variables of model reported 1
know origin (e.g., shedding rate) 0
state 0
degradation rate 0
transport 0