GENERON PMA95V VETfinder Real Time PCR Kits

Real-time PCR kits for PAP detection in feed according to the EURL-AP method

• Cat.# PMA95V VETfinder MBM RuminantsFREE RT-PCR kit (200 test)
• Cat.# PMA97V VETfinder MBM PorkFREE RT-PCR kit (200 test)
• Cat.# PMA98V VETfinder MBM PoultryFREE RT-PCR kit (200 test)

READ SAFETY INFORMATION AND DISCLAIMERS BEFORE USING THE KIT

Box Content

• reagents are supplied with an 5% extra volume. Generate Mastermix contains ROX as a passive reference dye

Reaction Set-Up
Protect reagents from light exposure as long as OLIGO Mix reagents are photosensitive.

Before Using

1. Leave the reagents to warm up at room temperature
2. Vortex briefly all the reagents
3. Spin (to avoid drops on the cap vials)

Prepare the VETfinder WORKING Mastermix by adding Diagenode Universal Mastermix tube and Diluent tube into the OLIGO Mix (See the workflow below). Then gently vortex the mix and spin briefly to obtain a single volume of VETfinder WORKING Mastermix for each tube.

This is just an example.
For the final layout of the plate, refer also to figure 2 (paragraph 5.4.1).

Remember: When setting the analysis, vortex briefly and spin the VETfinder WORKING Mastermix vial (to avoid drops on the cap vials) then transfer the aliquot of Mastermix and samples in the plate wells.

Introduction

Following EFSA’s positive opinion published on 22.09.2020, Regulation (EU) 2021/1372 has been in force and fully applicable in all Member States since 27 August 2021. The new legislation removes certain restrictions on the use of animal protein in feed by authorising the use of processed animal protein of porcine origin in poultry feed and processed animal protein of poultry origin in pig feed. The Regulation also allows the use of gelatine and collagen derived from ruminants in the feeding of non-ruminant farmed animals. The feeding ban on any farmed species with ruminant proteins (cattle, sheep, and goats) remains in force except milk and, now, collagen and gelatine. Feeding ruminants with any protein of animal origin is also completely prohibited, with some limited exceptions, such as milk. In addition, the prohibition of intraspecific recycling (so-called cannibalism) is strictly enforced in the Union.

In 2012, the European Union Reference Laboratory for Animal Protein in Feed (EURL AP) validated a new diagnostic method based on DNA analysis (PCR), which allows the detection of ruminant material potentially present in feed. Subsequently, in 2015 and 2018 respectively, EURL-AP also validated PCR methods that make it possible to detect the presence of material obtained from pigs or poultry in feed. They therefore make it possible to monitor the correct application of the ban on intra-specific recycling in pigs and poultry. The reference method for the detection of animal proteins in feed was originally defined in Annex VI of Commission Regulation n° 152/2009 then revised and amended by Commission Regulation n° 51/2013 and recently amended by Commission Implementation Regulation n° 1560/2020. The new regulation relies on the combination of light microscopy and real-time PCR for the detection of animal proteins in feed and on Standard Operating Protocols (SOPs) edited by the EURL-AP for technical implementation. These SOPs are binding and rely on the use of specific extraction kits, masters and calibrants. Generon developed a complete solution in full compliance with EURL-AP SOPs providing to the customer detection kits assembled using the reagents validated by EURL-AP. Detection kits are quality-controlled using the calibrants ERM provided by the JRC Geel. This document reflects and summarizes the indications of the EURL-AP SOP, however, please refer to the latest official version for a detailed method description.

Required materials

Plasmid calibrants (mandatory for SOP)

• ERM-AD482 ERM Ruminant pDNA Calibrant
• ERM-AD483 ERM Porcine pDNA Calibrant
• ERM-AD484K ERM Poultry pDNA Calibrant

Extraction kits and ancillary equipment for extraction (mandatory for SOP)

• EXD910 MagneSphere Stand for 1.5 ml tubes
• EXD911 MagneSphere Stand for 1.5 ml tubes
• EXD912 Wizard Mag DNA Purification kit (200 Prep)
• EXD913 Wizard Mag DNA Purification kit (400 Prep)

Matrix Reference Materials (mandatory for process controlling)

• PMA94F VETfinder heat treated meat powder (0.1% chicken and 0.1% bovine in 99.8% pig)
• PMA95F VETfinder heat treated meat powder (0.1% chicken in 99,9% pig)
• PMA97F VETfinder heat treated meat powder (0.1% bovine in 99,9% pig)
• PMA98F VETfinder heat treated meat powder (0.1% pig in 99,9% chicken)

Molecular biology lab commodities

• Absolute ethanol
• Bleach or any equivalent DNA-degrading reagent
• Isopropanol : 2-propanol p.a. grade
• Vortexer
• Pipette and filter tip sets
• 1.5 ml microtubes and related rack
• Microcentrifuge for 1.5 ml microtubes
• Real-Time PCR System with related software
• Optical Adhesive Seal or Optical Caps
• Optical reaction plate or Optical Tube Strips

DNA extraction

For DNA extraction follow the SOP of the EURL-AP as reported at the present link. This SOP is a binding complement to point 2.2.4. of Annex VI to Commission Regulation (EC) No 152/2009 as lastly amended by Commission Regulation (EU) No 51/2013 and describes the DNA extraction method to be used for the detection of animal DNA in feed samples as matrix. The SOP is based on two representative test portions of 100 mg per sample yielding two independent DNA extracts which can be used for PCR analysis. Controls to be included are also described, including one positive DNA extraction control made of a sample adulterated at a level of ≤ 0.1 % in mass fraction of PAP hence Generon VETfinder MRM (PMAxxF mentioned above) are fit for purpose. The extraction method is based on Promega Wizard® Magnetic DNA purification system. No alternative methods are allowed.

PCR platform cut-off setting

Before using the test for the detection of PAPs (PMA9xV series) in feed samples for routine analysis, the laboratory has to set the cut-off of its PCR platform(s). The PCR platform is considered as the combination of a thermocycler and the reagents used to perform a PCR. The platform is machine- specific and each cut-off values expressed in Ct established for one machine cannot be transferred to another machine without re-determining the cut-off value. When the laboratory receives a new batch of reagents, has to set the new PCR platform cut-off. The cut-off is a threshold value setting the difference between a positive result and a negative result. The cut-off of a PCR platform, eventually used for routine analysis, is determined by performing 16 calibrations with dedicated calibrants (plasmid calibrants) in 4 independent runs. For a detailed cut-off determination procedure follow the SOP of the EURL-AP as reported at the present link; we summarize it briefly below.

Fluorescence threshold
The Ct value and the cut-off value are relative parameters directly influenced by the level of the threshold. The baseline influences also the shape of the signal and the Ct calculated. For these reasons, it is mandatory to set the baseline and the threshold at the same value for all 4 plates used for cut-off determination (and during subsequent analyses of samples). The EURL-AP recommends to fix the baseline automatically and to set the threshold manually. Keep the same parameters for all plates (for the calibration of the platform but also for the runs of routine analysis).
Cut-off determination

Plate lay-out for cut-off determination
One calibration is made of 3 replicates from 3 calibrant levels (9 wells) but the calibration of a new PCR platform needs more data. Perform 4 runs and 4 calibrations per run as described in Figure 1. In the wells highlighted in green in Figure 1, the template DNA is made of the plasmid solution (calibrants are produced by JRC-Geel). Negative controls must be tested on each plate to check the absence of contamination.

In order to avoid repeated freeze/thaw cycles for the calibrants, the runs for the determination of the cut-off will be performed within 2 or 3 days. Once thawed, the calibrants shall be kept at 1 – 4 °C.

Remark: 16 calibrations spread on 4 runs is the minimum requirement to set the cut-off. However, when data are identified as outliers, they are removed from the calibration dataset. If the number of outliers is high (i.e. with more than 5% like loss of data from a complete calibration or from a run) additional calibrations or runs will be performed to replace removed data and to calculate an accurate cut-off.

Calculation of the cut-off (with the Excel file available on the EURL-AP website)
Each laboratory must download the spreadsheet available on the website of the EURL-AP (https://www.eurl.craw.eu/legal-sources-and-sops/method-of-reference- and-sops/ – EURL-AP SOP cut-off determination) and report the data of exact copy number values of specific calibrants batch and Ct values of the replicates from the 16 calibrations in the correct cells. A quality control of the cut-off value will be automatically provided by determining to which copy number the cut-off corresponds. The criterion to meet for this is:

• PMA95V – VETfinder MBM RuminantsFREE number of copies corresponding to the cut-off value > 9 copies.
• PMA97V – VETfinder MBM PorkFREE number of copies corresponding to the cut-off value > 3 copies.
• PMA98V – VETfinder MBM PoultryFREE number of copies corresponding to the cut-off value > 9 copies.

DNA Detection Assays

Reaction Set-Up
Before starting the practical work, edit the plate document. For general and more detailed instructions please refer to the user guide of the instrument and respective software version.

Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive. Before setting the analysis, we strongly advice to:

1. leave the reagents to warm up at room temperature
2. vortex briefly all the reagents
3. spin (to avoid drops on the cap vials)

Prepare VETfinder WORKING Mastermix (AWM) by adding the whole content of Diagenode Universal Mastermix and the whole content of DILUENT tubes into VETfinder OLIGO Mix tube . When setting the analysis, spin the VETfinder WORKING Mastermix vial (to avoid drops on the cap vials) then transfer the aliquot of WORKING Mastermix, DNA free WATER, and samples in the plate wells.

Reaction set-up for PMA95V (Ruminants) and PMA98V (Poultry)

1. Transfer 15 µL of AWM and 5 µL of DNA-free WATER into PCR plate wells according to the number of unknown samples, plus the number of wells acting as negative and positive Extraction and PCR control.
2. Add 5 µL of positive control into wells acting as a positive PCR control.
3. Add 5 µL of each sample to wells testing the unknown samples.
4. Add 5 µL of positive DNA extraction control (suitable for this use are our Matrix Reference Materials), mandatory for each DNA extraction series.
5. Add 5 µL of extraction blank control (negative extraction control), mandatory for each DNA extraction series.
6. Add 5 µL of negative control into wells acting as a negative PCR control.

Close wells and ensure no bubbles are present at the bottom of the wells.

Reaction set-up PMA97V (Pork)

1. Transfer 25 µL of AWM and 5 µL of DNA-free WATER into PCR plate wells according to the number of unknown samples, plus the number of wells acting as negative and positive Extraction and PCR control.
2. Add 5 µL of positive control into wells acting as a positive PCR control.
3. Add 5 µL of each sample to wells testing the unknown samples.
4. Add 5 µL of positive DNA extraction control (suitable for this use are our Matrix Reference Materials), mandatory for each DNA extraction series.
5. Add 5 µL of extraction blank control (negative extraction control), mandatory for each DNA extraction series.
6. Add 5 µL of negative control into wells acting as a negative PCR control.

Close wells and ensure no bubbles are present at the bottom of the wells.

Reaction set-up for inhibition control
When for the DNA samples the PCR does not deliver any positive results, the laboratory must perform and check the inhibition control as follows (see also 6.1).

TEST CONDITION

• Inhibition control alone: one well with 15 µL (or 25 µL) of AWM + 5 µL of DNA-free WATER + 5 µL of plasmid calibrant at the lowest level of copies (instead of the sample);
• Inhibition control alone + DNA sample: one well with 15 µL of AWM + 5 µL of plasmid calibrant at the lowest level of copies (instead of the DDNA-free-water) + 5 µL of sample for which inhibition occurs (is possible to test different dilutions of the sample)

Detector set-up

• Target
  

  • Ruminant Poultry or Pork DNA

• Reporter Dye
  

  • FAM

• Quencher Dye

  • BHQ1-NFQ

Thermal Cycling Conditions

Cycling set-up for PMA95V (Ruminants)

Reaction set-up PMA97V (Pork) and PMA98V (Poultry)

PCR plate layout for sample analysis
PCR shall be performed on at least two different dilutions of the DNA extract of a test portion and each sample is analyzed with two replicate test portions. The EURL-AP recommends testing first the undiluted extract and a 10-fold diluted extract.

Each plate shall contain per target under analysis :

• at least one well with a positive DNA target control (also called positive PCR control)
• at least one well with an amplification reagent control (also called no template control or negative PCR control)

For each DNA extraction series, it is mandatory to analyze at least one plate in at least one well per target under consideration :

• the extraction blank control (also called negative extraction control)
• the positive DNA extraction (control containing a 0.1% mass fraction of PAP or lower, e.g Generon Cat# PMAxxF). The content of the positive DNA extraction control should be close to the limit of detection of the method.

Plate preparation
The layout of the plate is defined before PCR begins. An example realized with the Excel software is presented in Figure 2.

The real-time PCR mix prepared as described at point 5.1 is pipetted into the wells. Samples to be tested (DNA extract, control, or water) are added afterward in their respective wells.

Results and Data Interpretation

The results interpretation must be by what is reported in the specific EURL-AP SOP, and can only be done once the PCR step is completed. Before analyzing any PCR result for a defined target on a sample the expected results for both extraction and both PCR controls must be correct, otherwise, the PCR run has to be repeated. If the problem remains, then perform new extractions or new PCR runs using new reagents. Hence:

Each sample is analyzed using two replicate test portions and two PCR to be run per test portion on two different dilutions of the DNA extract. If a PCR result is positive at least for one of the dilutions, the test portion will be considered as positive (Figure 3).

However, if both PCR results are negative (Figure 4), it is not possible to conclude directly that the test portion is negative and it is necessary to provide evidence that the absence of the signal is not due to total PCR inhibition. This can be done via the use of an inhibition control or by obtaining a positive PCR result on the extracts of the test portion with another target (e.g. with a plant universal target or another animal target ).

In any case, if there are indications of inhibition, the operator shall repeat the PCR at other dilution rates (e.g. 3x, 20x, 30x), and if for one of these other dilution rates, the test portion delivers a positive result, the test portion has to be considered as positive. The final interpretation of results for a sample with a PCR target shall take into consideration the two determinations performed i.e. PCR results on both test portions (Figure 5), hence:

1. If both test portions are positive with a PCR target the sample is positive for that target.
2. If both test portions are negative with a PCR target, then the sample has to be considered as negative for that PCR target after it has been demonstrated that there is no PCR inhibition.
3. If total PCR inhibition is demonstrated, an additional purification of the DNA extract must be performed.
4. If results from the two test portions are inconsistent, the genetic amplification for the target under consideration shall be repeated (once again two dilutions per test portion). If by doing so both results are consistent, it is that result that becomes the final PCR result for the target under consideration, while if results on both test portions remain conflicting, then the result will be considered negative.
5. If however the laboratory suspects that the DNA extracts can be the cause of the inconsistency (e.g. a too large difference between Ct’s on both test portions analyzed at a same dilution rate – more than 3 Ct units), a new DNA extraction and the subsequent genetic amplification shall be performed on two test portions before interpreting the results.

The following table represents the interpretation of the final result:

Inhibition control
When a given test portion does not deliver positive results with any of the tested PCR targets, then PCR inhibition has to be checked. Two options are possible, one of them being the use of an inhibition control. Procedure to follow:
A known amount of target not exceeding 100 copies per well, e.g. the ERM plasmid calibrant at the lowest level of copies, shall be analyzed in two different conditions (each at least in one well):

1. Without any other DNA source than the added target
2. In the presence of the DNA extract (different dilutions may be tested), it is analyzed for inhibition.

For the reaction set-up refer to paragraphs 5.1.3. To interpret the results of the inhibition control (Figure 5), the PCR must be positive with the test done in conditions #1. Should it be negative in these conditions, then the test is not valid. On the other hand, if a positive result is obtained in condition

2, this means there is no inhibition (or at least no total inhibition) while

if it is a negative result this means that the absence of amplification results from an inhibitory effect on PCR of the DNA extract at the dilution at which it was tested.

Validation of the product

The assay was validated on Bio-Rad CFX and it is compatible with Bio-Rad MiniOpticon, MyGO mini, MyGO Pro, bCube and Applied BioSystems 7500 fast, Step-one and Step-one plus, Quantstudio 5-7 Thermo, Light Cycler 480 Roche, Mx3000P and Mx3005P Stratagene, Rotor-Gene Q Qiagen, Agilent AriaMx RT- PCR System. The assay is not compatible with Roche Light Cycler I and II. Inquire our tech support (technical.support@generon.it) for details.

Detection limit
According to the results obtained during the assessment of the PCR step on 4 PCR platforms by the EURL AP, the cut-off calculated for the specific targets is:

• Ruminants cut-off at 15 copies;
• Poultry cut-off at 15 copies;
• Pork cut-off at 5 copies;

Specificity
The specificity of the primers was tested both in silico through comparisons with the NCBI sequence database, and experimentally on a collection of reference DNAs. For the results refer to the specific EURL-AP SOP.

Storage & Expiry information

Expiry date: see date on the packaging, product validity refers to the product kept intact in its original packaging. Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive. Store frozen. Despite the day of actual use of the kit components and their mixing, all the reagents are considered expired on the date indicated on the Kit box.
Avoid repeated thawing and freezing (>4x) and in case subaliquot the working mastermix.

Troubleshooting

Concomitant no target nor IAC amplification, or amplification plots grossly abnormal. Possible causes and corrective actions:

• An excess of DNA and/or PCR inhibitors inhibits the reaction
• Inadequate sealing of optical caps/film caused sample evaporation. Redo the analysis using proper tools and proper optical caps/film to secure perfect sealing.
• Inadequate consumables. Redo the analysis and use only optical grade 96-well plates and optical adhesive seal or optical 8-well strips and caps.
• Positive Control reactions failed to amplify: Positive Control DNA was not added to the reaction wells. If other reactions look normal, there may be no need to repeat the run.
• Negative Control reactions are positive: Contamination of the negative control vial or the PCR mix with positive DNA. Use more care to prevent contamination while handling assay reagents and setting up assays.
• (Please, be aware that) the intensity of the fluorescence signal is influenced by the type of disposable PCR labware: well plates, tubes, strip tubes, adhesive films, caps, cap strips. On Bio-Rad CFX it is recommended the use of white tubes/plates.

For any request of support contact Generon at: technical.support@generon.it

Disclaimers

General S.p.A. warrants the products will be free of defects in materials and workmanship when used under the instructions and before the expiration date marked on the product packaging under the storage conditions recommended in the instructions and/or on the package. Application protocols published by Generon are intended to be only guidelines for the buyers of the products. Each buyer is expected to validate the applicability to his application. General makes no other warranty, expressed or implied. There is no warranty of merchantability or fitness for a particular purpose. Generon’s sole obligation concerning the foregoing warranties shall be, at its option, to either replace or to refund the purchase price of the product(s) or part thereof that proves defective in materials or workmanship within the warranty period, provided the customer notifies Generon promptly of any such defect. General shall not be liable for any direct, indirect or consequential damages resulting from economic loss or property damages sustained by the buyer or any customer from the use of the product(s).

Generon S.p.A.
Via San Geminiano, 4 41030 San Prospero (MO)- Italia

• +39 059 8637161
• +39 059 7353024
• technical.support@generon.it
• www.generon.it

References

• Kit Analisi Alimenti per Ricerca Batteri, Virus e Contaminanti - Generon
• Legal sources and SOPs – EURL-AP

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