qPCR
Basic information
Latin name | Anaxyrus boreas, A. canorus, A. exsul, A. nelsoni |
Common name | Western Toad Complex |
Broad taxonomic group | amphibian |
eDNA source | water |
assay type | qPCR |
5′ reporter dye (e.g. FAM, VIC, TET, NED) | FAM |
TaqMan MGBNFQ (minor groove binder non fluorescent quencher) | yes |
PCR chemistry | TaqMan Environmental 2.0 Master Mix (Life Technologies) |
latest publication year | 2018 |
Number of citations | 1 |
Citation | 93 |
Assay #129
gene / region | Cytochrome c oxidase subunit I |
primer F (5′ – 3′) | CAAGAAAGAACCATTYGGGTACATG |
primer R (5′ – 3′) | TCGACATTAAGATCTGTCGTAAATATGTG |
probe (5′-3′) | AATGTCTATTGGTCTTCTAGGGT |
Validation score and minimum criteria
Validation level |
4 |
|
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 | 1 |
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 | 1 |
closely related | 1 |
co-occurring | 1 |
target tissue | 1 |
tissue source | 0 |
geographic scope | 1 |
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 | 1 |
how many per species | 1 |
geographic origin | 1 |
tissue source (fresh or old) | 1 |
non-target amplification checked via sequencing | 0 |
DNA concentration | 0 |
basic geographic scope | 1 |
unidirectional lab flow | 0 |
extensive geographic testing | 0 |
method of extraction | 1 |
modifications from established protocol | 1 |
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 | 0 |
precipitation chemicals added | NA |
centrifugation force and time for precipitation | NA |
temperature of precipitation | NA |
storage between collection and processing | 0 |
“field” blanks | 0 |
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 | 0 |
determined limit of detection | 1 |
method of establishment | 1 |
multiple replicates of standard curve | 1 |
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 | 0 |
environmental gradients (e.g., abiotic biotic conditions) | 0 |
different known target species densities | 0 |
testing against data from traditional methods | 0 |
inhibition testing | 1 |
geographic region tested | 1 |
source of species occurrence information | 0 |
multiple samples per species | 1 |
geographic origin of tissue samples | 1 |
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 | 1 |
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 |
Assay #130
gene / region | Cytochrome b |
primer F (5′ – 3′) | CTACAAAGACATCTTCGGCTTCG |
primer R (5′ – 3′) | AGTTGTCGGGGTCACCCAA |
probe (5′ – 3′) | ACTAATACTAGCCCTTCTAGCC |
Validation score and minimum criteria
Validation level |
4 |
|
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 | 1 |
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 | 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 | 1 |
closely related | 1 |
co-occurring | 1 |
target tissue | 1 |
tissue source | 0 |
geographic scope | 1 |
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 | 1 |
how many per species | 1 |
geographic origin | 1 |
tissue source (fresh or old) | 1 |
non-target amplification checked via sequencing | 0 |
DNA concentration | 0 |
basic geographic scope | 1 |
unidirectional lab flow | 0 |
extensive geographic testing | 0 |
method of extraction | 1 |
modifications from established protocol | 1 |
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 | 0 |
precipitation chemicals added | NA |
centrifugation force and time for precipitation | NA |
temperature of precipitation | NA |
storage between collection and processing | 0 |
“field” blanks | 0 |
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 | 0 |
determined limit of detection | 1 |
method of establishment | 1 |
multiple replicates of standard curve | 1 |
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 | 0 |
environmental gradients (e.g., abiotic biotic conditions) | 0 |
different known target species densities | 0 |
testing against data from traditional methods | 0 |
inhibition testing | 1 |
geographic region tested | 1 |
source of species occurrence information | 0 |
multiple samples per species | 1 |
geographic origin of tissue samples | 1 |
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 | 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 |