TITLE: qPCR assay for detecting round goby invasive fish species Neogobius melanostomus
AUTHORS: Omneya Ahmed, Alexander Eiler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Members of Ponto-Caspian gobies are predatory fish which have colonized freshwaters and brackish waters in Europe and North America causing a lot changes in the native ecosystems.</div><div class = "text-block">Several eDNA assays were developed to identify round goby fish. We are going to describe two successful assays.</div></div>

[STEPS]
?. [DNA extraction]
A tissue of round goby was extracted with Qiagen DNeasy blood and tissue extraction kithttps://www.qiagen.com/us/shop/pcr/dneasy-blood-and-tissue-kit/The quality of DNA and 260/280 ratio were checked by NanoDrop instrument for nucleic acid measurements.
?. [DNA extraction]
PrimersNeogobius melanostomusPrimerReferenceTemperatureLengthGC contentNeo_Mel_COI_F015’-CTTCTRGCCTCCTCTGGWGTTG-3’Andersen et al., 201659.6 -62.82254-59Neo_Mel_COI_R015’-CCCWAGAATTGASGARATKCCGG-3’58.9 -63.92347-56Neo_Mel_COI_P015’-FAM-CAGGCAACTTRGCACATGCAG-BHQ-3’60.1 - 62.92152-57NeoMeI_IK_F15’- TATGTGATGATCGGACAGC-3’Adrian-Kalchhauser & Burkhardt-Holm, 2016.1953-56NeoMel_IK_R15’- GTTCTCTAGTCAGCTCGCT-3’1945-51NeoMeI_IK_Probe5’-FAM-CATCTTTCTCGGCTTATTCCCCA-BHQ-3’23
Neogobius melanostomusPrimerReferenceTemperatureLengthGC contentNeo_Mel_COI_F015’-CTTCTRGCCTCCTCTGGWGTTG-3’Andersen et al., 201659.6 -62.82254-59Neo_Mel_COI_R015’-CCCWAGAATTGASGARATKCCGG-3’58.9 -63.92347-56Neo_Mel_COI_P015’-FAM-CAGGCAACTTRGCACATGCAG-BHQ-3’60.1 - 62.92152-57NeoMeI_IK_F15’- TATGTGATGATCGGACAGC-3’Adrian-Kalchhauser & Burkhardt-Holm, 2016.1953-56NeoMel_IK_R15’- GTTCTCTAGTCAGCTCGCT-3’1945-51NeoMeI_IK_Probe5’-FAM-CATCTTTCTCGGCTTATTCCCCA-BHQ-3’23
?. [DNA extraction]
Standard DNA dilutionTo determine both limit of quantification and limit of detection, genomic DNA of round goby was serially diluted from 1e2-1e-4 .
2 µl
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The composition of PCR mixturePCR reagentStock solutionWorking solutionFinal concetration  
      (μL)TaqMan Environmental Mastermix 22X1X10Forward primer10 μM0.4 μM1Reverse primer10 μM0.4 μM1TaqMan probe2.5 μM0.1 μM1Internal control (IC) primer/probe mix1IC-DNA0.5DNase/RNase free water8.5Template2Total volume25
PCR reagentStock solutionWorking solutionFinal concetration  
      (μL)TaqMan Environmental Mastermix 22X1X10Forward primer10 μM0.4 μM1Reverse primer10 μM0.4 μM1TaqMan probe2.5 μM0.1 μM1Internal control (IC) primer/probe mix1IC-DNA0.5DNase/RNase free water8.5Template2Total volume25
?. [DNA extraction]
Amplification conditionsStepTimeTemp50 cyclesEnzyme activation10 min95Denaturation15 s95Data collection1 min60
0 °C
StepTimeTemp50 cyclesEnzyme activation10 min95Denaturation15 s95Data collection1 min60
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QPCR was performed in BioRad qPCR machine CFX96.
Analysis of qPCR results was performed through CFX maestro softwarehttps://www.bio-rad.com/en-se/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments?ID=OKZP7E15
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The analysis of PCR internal controlThe Cq value obtained with the internal control will vary significantly depending on the extraction efficiency, the quantity of DNA added to the PCR reaction and the individual machine settings. Cq values of 26 or 27±3 are within the normal range.
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To check for inhibitors in DNA samples, internal control was used, http://www.primerdesign.co.uk/assets/files/internal_control_handbook_dna.pdf?timestamp=1469446474OR: https://www.thermofisher.com/order/catalog/product/4308321.