TITLE: qPCR assay for detecting Batrachochytrium dendrobatidis
AUTHORS: Omneya Ahmed Osman, Johan Andersson, Alexander Eiler, Mats Töpel, Tomas Larsson, Tomas Larsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The fungus </span><span style = "font-style:italic;">Batrachochytrium dendrobatidis</span><span> (Bd) was first detected in Norway in 2017, and thus indicate the arrival of an invasive black-listed species in the country. 
Here we report the details of real time PCR assay which was used to screen for</span><span style = "font-style:italic;"> B. dendrobatidis</span><span> from  water samples collected from different locations in Norway.




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

[STEPS]
?. [DNA extraction]
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 kit: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en
?. [DNA extraction]
Internal DNA extraction control 
When performing DNA extraction, it is often advantageous to have an exogenous source of DNA template that is spiked into the lysis buffer. This control DNA is then co-purified with the sample DNA and can be detected as a positive control for the extraction process. Successful co-purification and qPCR for the control DNA also indicates that PCR inhibitors are not present at a high concentration.
?. [DNA extraction]
Real Time PCR A B.dendrobatidis specific primer and probe mix is provided by Primerdesign Ltd which was detected through the FAM channel. The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR amplification, forward and reverse primers hybridize to the B.dendrobatidis DNA. A fluorogenic probe is included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected on a range of qPCR platforms.  AB1ComponentResuspend in water2								B.dendrobatidis primer/probe mix (BROWN)

													165 μl

					3								Internal extraction control primer/probe mix (BROWN)

													165 μl
AB1ComponentResuspend in water2								B.dendrobatidis primer/probe mix (BROWN)

													165 μl

					3								Internal extraction control primer/probe mix (BROWN)

													165 μl
?. [DNA extraction]
Positive and internal controlsResuspend the internal control template and positive control template in the template preparation buffer supplied, according to the table below:
  AB1Component (heat-sealed foil)Resuspend in template preparation buffer2					3								Internal extraction control DNA (BLUE)

													600 μl

					4B.dendrobatidis Positive Control Template (RED) *								500 μl
* This component contains high copy number template and is a VERY significant contamination risk. It must be opened and handled in a separate laboratory environment, away from the other components.
AB1Component (heat-sealed foil)Resuspend in template preparation buffer2					3								Internal extraction control DNA (BLUE)

													600 μl

					4B.dendrobatidis Positive Control Template (RED) *								500 μl
?. [Preparation of standard curve dilution series]
1) Pipette 90 μl of template preparation buffer into 8 tubes and label them.2) Pipette 10 μl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 10 μl from tube 2 into tube 3 
5) Vortex thoroughly 
Repeat steps 4 and 5 to complete the dilution series 
   AB1Positive controlCopy number2									Tube 1 Positive control (RED)

						1e6 per 5 μl3									Tube 2

						1e5 per 5 μl4									Tube 3

						1e4 per 5μl5									Tube 4

						1 e3 per 5 μl6									Tube 5

						1e2 per 5 μl7									Tube 6

						1e1 per 5 μl8Tube 6.55 per 5 μl9Tube 71e-1per 5 μl10Tube 81e-2 per 5 μl
A standard with very low copy number (theoretically 0.1 cells per PCR reaction) was prepared to determine limit of detection of this assay.
AB1Positive controlCopy number2									Tube 1 Positive control (RED)

						1e6 per 5 μl3									Tube 2

						1e5 per 5 μl4									Tube 3

						1e4 per 5μl5									Tube 4

						1 e3 per 5 μl6									Tube 5

						1e2 per 5 μl7									Tube 6

						1e1 per 5 μl8Tube 6.55 per 5 μl9Tube 71e-1per 5 μl10Tube 81e-2 per 5 μl
?. [PCR program for BD detection]
ABC1Componentμlμl2SsoAdvanced Universal Probes
  Supermix&nbsp;1053B.dendrobatidis primer/probe mix
  (BROWN)&nbsp;10,54RNase/DNase free water
  (WHITE)&nbsp;41,55Final Volume&nbsp;157Pipette 5 μl DNA template if the total PCR mixture is 20 μl or 3 μl of DNA template if the total PCR mixture is 10 μl.For negative control wells, we used 5 or 3 μl of RNase/DNase free water.
ABC1Componentμlμl2SsoAdvanced Universal Probes
  Supermix&nbsp;1053B.dendrobatidis primer/probe mix
  (BROWN)&nbsp;10,54RNase/DNase free water
  (WHITE)&nbsp;41,55Final Volume&nbsp;157
?. [PCR program ]
ABCD1 Step&nbsp;Time&nbsp;Temp (C)2Enzyme activation&nbsp;2 min&nbsp;953Cycling x50&nbsp;Denaturation&nbsp;10 s&nbsp;954DATA COLLECTION *&nbsp;1min60
ABCD1 Step&nbsp;Time&nbsp;Temp (C)2Enzyme activation&nbsp;2 min&nbsp;953Cycling x50&nbsp;Denaturation&nbsp;10 s&nbsp;954DATA COLLECTION *&nbsp;1min60
?. [PCR for internal control]
AB1								Component Volume

					Volume (μl)2SsoAdvanced Universal Probes
  Supermix103								Internal extraction control primer/probe mix (BROWN)

					14								RNase/DNase free water (WHITE)

					35								Final Volume

					15Pipette  5 μl of DNA template to reach 20 μl final volume in each well.For negative control wells use 5 of RNase/DNase free water.
AB1								Component Volume

					Volume (μl)2SsoAdvanced Universal Probes
  Supermix103								Internal extraction control primer/probe mix (BROWN)

					14								RNase/DNase free water (WHITE)

					35								Final Volume

					15
?. [Interpretation of results]
ABCDE1									Target
(FAM)

						Internal
control
(VIC)									Positive
control

															Negative
control

															Interpretation

						2									≤ 30

															+/ -

															+

															-

															POSITIVE QUANTITATIVE RESULT

									calculate copy number

						3									&gt; 30

															+

															+

															-

															POSITIVE QUANTITATIVE RESULT

									calculate copy number

						4									&gt; 30

															-

															+

															-

															POSITIVE QUALITATIVE RESULT

									do not report copy number as this
may be due to poor sample extraction

						5									-

															+

															+

															-

															NEGATIVE RESULT

						6									+ /-

															+/ -

															+

															≤ 35

															EXPERIMENT FAILED

									due to test contamination

						7									+ /-

															+/ -

															+

															&gt; 35

															*

						8									-

															-

															+

															-

															SAMPLE PREPARATION FAILED

						9									+ /-

															+/ -

															-

															+ /-

															EXPERIMENT FAILED
Quantitation cycle (Cq)Positive control template, 1e6 per 5 μl,  is expected to amplify between Cq 16 and 23. Failure to satisfy this quality control criterion is a strong indication that the experiment has been compromised. Where the test sample is positive and the negative control is positive with a Cq > 35, the sample must be reinterpreted based on the relative signal strength of the two results: 


Internal PCR control 
The 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 28±3 are within the normal range. When amplifying a B. dendrobatidis sample with a high genome copy number, the internal extraction control may not produce an amplification plot. This does not invalidate the test and should be interpreted as a positive experimental result.
ABCDE1									Target
(FAM)

						Internal
control
(VIC)									Positive
control

															Negative
control

															Interpretation

						2									≤ 30

															+/ -

															+

															-

															POSITIVE QUANTITATIVE RESULT

									calculate copy number

						3									&gt; 30

															+

															+

															-

															POSITIVE QUANTITATIVE RESULT

									calculate copy number

						4									&gt; 30

															-

															+

															-

															POSITIVE QUALITATIVE RESULT

									do not report copy number as this
may be due to poor sample extraction

						5									-

															+

															+

															-

															NEGATIVE RESULT

						6									+ /-

															+/ -

															+

															≤ 35

															EXPERIMENT FAILED

									due to test contamination

						7									+ /-

															+/ -

															+

															&gt; 35

															*

						8									-

															-

															+

															-

															SAMPLE PREPARATION FAILED

						9									+ /-

															+/ -

															-

															+ /-

															EXPERIMENT FAILED
?. [PCR for internal control]
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=OKZP7E15
?. [DNA extraction]
Internal control  of the internal control were mixed with lysis buffer as given in the extraction protocol. Purification and amplification of the internal control DNA was tested as well as the presence of PCR inhibitors. The primers are present at PCR limiting concentrations which allows multiplexing with the target sequence primers. Amplification of the control DNA does not interfere with the detection of the B. dendrobatidis target DNA even when present at low copy number.
4 µl