TITLE: The detection of freshwater pearl mussel 
AUTHORS: Omneya Ahmed Osman, Alexander Eiler, Mats Töpel, Tomas larsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>qPCR assay to detect freshwater pearl mussels </span><span style = "font-style:italic;">Margaritifera margaritifera</span><span> in order to monitor populations in their natural environment. 



</span></div></div>

[STEPS]
?. [Primers]
Stoeckle et al., 2016 designed primers targeting 16S rRNA gene of Margartifera margaritifera.The sequence of the primer set is:MarMa_16S2.1: 5’- GCAACACGGAAAACCCC TG -3’MarMa_16S1.2: 5’- GGCT GCGCTCATGTGAATTA -3’.
?. [PCR reaction]
A master mix was prepared and calculated according to the number of standard dilutions, samples and negative controls to run. The PCR mixture per sample was as follows:  ABCD1Reagentworking solfinal concµl2SsoAdvanced
  Universal SYBR® Green Supermix2x1x103MarMa_16SF
  primer10 uM0,25 uM0,54MarMa_16SR
  primer10 uM0,25 uM0,55DEPC-water  66DNA template  37∑  208Sample or qPCR plate should be carefully sealed with no marking on the tube to facilitate plate reader step.
ABCD1Reagentworking solfinal concµl2SsoAdvanced
  Universal SYBR® Green Supermix2x1x103MarMa_16SF
  primer10 uM0,25 uM0,54MarMa_16SR
  primer10 uM0,25 uM0,55DEPC-water  66DNA template  37∑  208
?. [Primers]
The concentration of freshwater pearl mussel standard was measured by PicoGreen assay (http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf) before preparing standard serial dilutions.Serial dilutions, 10, 1, 0.1, 0.01 and 0.001 ng, were prepared to generate standard curve in each qPCR run.Unio pictorum or Äkta målarmussla was used to test the specificity of qPCR detection.
?. [PCR program]
ABCD1Amplification stepTime&nbsp;Temp&nbsp;23&nbsp;Enzyme activation15 sec98&nbsp; °C&nbsp;4Denaturation&nbsp;10 sec98 °C&nbsp;40 cycles5Annealing10 sec60 °C&nbsp;6Extension20 sec72 °C&nbsp;7Melt curve analysis 63 to 99°C&nbsp;
ABCD1Amplification stepTime&nbsp;Temp&nbsp;23&nbsp;Enzyme activation15 sec98&nbsp; °C&nbsp;4Denaturation&nbsp;10 sec98 °C&nbsp;40 cycles5Annealing10 sec60 °C&nbsp;6Extension20 sec72 °C&nbsp;7Melt curve analysis 63 to 99°C&nbsp;
?. [Plate setup]
Setup and design your qPCR plate  in the template of qPCR machine before preparing your qPCR run. At least 3 or 4 replica of standard dilutions should be included in each run.Three replica of each sample was performed.
?. [PCR program]
QPCR was performed in BioRad qPCR machine CFX96.Analysis of the results was done by CFX maestro softwarehttps://www.bio-rad.com/en-se/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments?ID=OKZP7E15QPCR efficiency, limit of detection and quantification range of standard were checked for each run.Results can be evaluated through data analysis windows where there is a quantification, standard melt curve and  RFU spreadsheet.
?. DNA extraction was performed using Qiagen DNeasy power water sterivex kit. The quality of the extracted DNA was estimated using Nanodrop. 

Qiagen DNeasy power water sterivex protocol can be found in this link: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en