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Systematic and massive detection of SARS-CoV-2 with PCR

ADNucleis aims is to standardized the use of real time PCR through the help of an affordable cost of reagents and tool.

That’s why focus our efforts for 15 years to create systems which bring real time PCR accessible for most laboratories on the planet.

SEQUENCE automate is now compatible with our Coronavirus (SARS-CoV-2) detection kit, working directly form saliva sample, inside a whole range of detection including influenza and respiratory virus.

SEQUENCE follows point-of-care diagnostic strategy for every medical structures and laboratories, at an affordable cost.

    About ADNucleis

    Founded in 2007 by Pr Michel FRANCK, ADNucleis is a spin-off of the Veterinary School of Lyon and is specialized in the conception of diagnostics solutions. Focused on developing affordable in-vitro diagnostic kit for Human Health, Animal health, and Food quality, ADNucleis is positioned with a brand diagnostic PCR reagents kits solution compatible with most of the PCR systems on the market.

    Some of our main detection kits:

    Human health : Coronavirus, Influenza, respiratory virus, Lyme, CBEU, Antibiotics resistance

    Animal Health : PRRSV, Mastitis, QFever, Lyme…

    Food quality : Hygien indicators including E.coli, pseudomonas, total flora…

    Supported by the Ministry of Research (every year since 2007), Europe in 2012 (Susclean project), the Ministry of Industry (since 2014), the Auvergne Rhône-Alpes Region (since 2015) and BPI France (since 2008 via Oséo), ADNucleis never stopped to innovate by making real time PCR an affordable and easy-to-use technical.

    contact@adnucleis.com

    PRRS NA/HP detection kit Introduction

    Instruction Manual

    Reverse transcriptase Polymerase Chain Reaction (RT-PCR) in real time for the qualitative detection of

    North America Porcine Reproductive and Respiratory Syndrome virus

    KIT HQS_PRRS-NA_i_tqm with IPC

    48/96 reactions

    The kit include and internal positive control

    The kit uses the Taqman® probe technology to measure DNA/RNA amplification

    Storage conditions:

    Keep all reagents at -20 °C until use and after the first use.

    Manufacturer
    ADNUCLEIS SAS
    Siret 497 802 165 000 17
    TVA intraCEE : FR 3649780216500017
    * 3 Route des Pierres Blanches
    F- 69290 GREZIEU LA VARENNE
    FRANCE
    Pages: 1 2

    RT-PCR kit content-storage

    rev 1.0 (13/07/2016)

    IV.               Description and content of the kit

    The RT-PCR kit is ready to use for the specific detection of a target. The kit contains reagents and enzymes necessary of the amplification of a specific target, synthetic Target positif RNA (positive control) and of RNA inhibitory control (IPC) (see table 1). Fluorescence is emitted and measured individually by an optical system during PCR. The detection of the amplified fragment is usually carried out by the fluorimeter using the channels listed in table 2.

    Table 1:

    Components of the kits 48 reactions 96 reactions Reconstitution
    Volume (µl) Volume (µl)
    PCR pre-mix
    		 1160 2000 Ready to use
    Taq polymerase Nucleis (Taq N) 116 200 Ready to use
    Reverse transciptase Nucleis (RT N) 58 100 Ready to use
    Target RNA Positive control 200 200 Ready to use
    RNA IPC (inhibitory control) 150 300 Ready to use

    Table 2: Fluorophores usually used in duplex target-IPC kits.

    Target Fluorophore Excitation Emission
    Target FAM 499 519
    IPC VIC 520 548

    (Please check the kit certificat for the fluorophore used in a specific kit.)

    Alternatives:

    -channel FAM: (ABI Systems, smartcycler II; Chromo 4/CFX96, System Agilent MX and aria, realplex), channel 530 (LC480), channel Green (RotorGene)

    -channel VIC: Channel VIC (ABI systems, Biorad CFX96, Agilent Mx and aria, realplex), channel Alexa 532 (Smart cyclerII), Channel 560 (LC 480); channel Yellow  (RotorGene)

     

    Required material and reagents not provided

    • Elution buffer
    • A RNA extraction and purification kit
    • Biological hood
    • qPCR instrument
    • Micro centrifuge
    • Vortex
    • Plates / tubes for qPCR
    • Micropipettes
    • Filtre tips for micropipettes
    • Sterile microtubes
    • A viral RNA extraction and purification kit
    • Non powdered gloves

    V.               Storage

    All reagents must be stored at -20°C. Storage at 4°C is forbidden.

    All reagents can be used until the expiration date indicated on the kit

    Many freezing/thawing cycles (>3x) must be avoided, could lead to decrease in sensitivity

    VI.               Cautions and notes

    • Read carefully instructions before starting.
    • The experiment must be performed by competent staff
    • Instruments must have been properly installed, calibrated and maintained according to the manufacturer’s recommendations
    • Clinical samples are potentially infectious and must be processed under a laminar flow hood/
    • The experiment must be performed according to good laboratory practices
    • Do not use this kit after expiration date
    • Many freezing/thawing cycles of the reagents must be avoided, and could lead to decrease of sensitivity
    • Once defrosted, spin down briefly the tubes before use
    • Spin down briefly enzymes tubes to pellet the viscous content at the bottom of the tubes.
    • Use of ice or cooling block is mandatory to prepare the mix and dispense reconstituted mix in wells.
    • Define 3 working areas: 1)Isolation of DNA/RNA, 2) Preparation of the reaction mix and 3) Amplification/detection of amplified products.
    • Pipettes, reagents and other materials must not cross each area
    • Specific caution is required to preserve the purity of the reagents and reaction mixtures. The use of gloves is mandatory. Appropriate methods of preparation of DNA should be used.
    • Always use filtered tips for micropipettes
    • Use specific lab coat and gloves in each working area
    • Do not pipette with mouth and do not eat, drink or smoke in the area
    • Avoid sprays

    VII.               Collection of samples, transport and storage

    • Collect samples in sterile tubes
    • The samples should be extracted immediately or frozen from -20°C to 80°C
    • Transport of clinical samples must obey local regulations for this type of infectious agents.

    real Time RT-PCR principles-taqman

    III.               Principle of the detection

    The ADNUCLEIS RT-PCR kits are using reverse transcription to synthesize RNA into cDNA followed by the amplification of the cDNA by real time RT- PCR. Kits may also include  inhibition control (IPC) made of synthetic RNA. This control ensures that a negative result may not be due to the presence of PCR inhibitors at high quantity.

    The test is performed from the RNA extracted from the sample. For taqman duplex kits with IPC, the target is usually detected by  the FAM channel and the IPC by the JOE/VIC channel (color Probe may be different, please refer to the kit certificat for further information). All emit at a specific fluorescence following its hydrolysis during the elongation of the amplification product. The measurement of the intensities of the real time fluorescence relates the accumulation of specific amplification products.

    This amplification system has been validated on:

    • A synthetic RNA produced in vitro from a plasmid containing a specific insert of the target or of the IPC.

    IV.               Description and content of the kit

    The PRRS NA virus real time RT-PCR kit is ready to use for the specific detection of the type 2 (NA/HP) PRRS virus. The kit contains reagents and enzymes necessary of the amplification of type 2 (NA/HP) PRRS virus, synthetic PRRS NA RNA (positive control) and of RNA control (IPC) of RT-PCR inhibition (see table 1). Fluorescence is emitted and measured individually by an optical system during PCR. The detection of the amplified fragment is carried out by the fluorimeter using the channels listed in table 2.

     

    Table 1:

    Components of the kits 48 reactions 96 reactions Reconstitution
    Volume (µl) Volume (µl)
    PCR pre-mix 1160 2000 Ready to use
    Taq polymerase Nucleis (Taq N) 116 200 Ready to use
    Reverse transciptase Nucleis (RT N) 58 100 Ready to use
    PRRS NA RNA Positive control 200 200 Ready to use
    RNA IPC (inhibitory control) 150 300 Ready to use

     

    Table 2

    Target Fluorophore Excitation Emission
    PRRS NA virus FAM 499 519
    IPC VIC 520 548

     

    Alternatives:

    -channel FAM: (ABI Systems, smartcycler II; Chromo 4/CFX96, System Agilent MX and aria, realplex), channel 530 (LC480), channel Green (RotorGene)

    -channel VIC: Channel VIC (ABI systems, Biorad CFX96, Agilent Mx and aria, realplex), channel Alexa 532 (Smart cyclerII), Channel 560 (LC 480); channel Yellow  (RotorGene)

     

    Required material and reagents not provided

    • Elution buffer
    • A RNA extraction and purification kit
    • Biological hood
    • qPCR instrument
    • Micro centrifuge
    • Vortex
    • Plates / tubes for qPCR
    • Micropipettes
    • Filtre tips for micropipettes
    • Sterile microtubes
    • A viral RNA extraction and purification kit
    • Non powdered gloves

    V.               Storage

    All reagents must be stored at -20°C. Storage at 4°C is forbidden.

    All reagents can be used until the expiration date indicated on the kit

    Many freezing/thawing cycles (>3x) must be avoided, could lead to decrease in sensitivity

    VI.               Cautions and notes

    • Read carefully instructions before starting.
    • The experiment must be performed by competent staff
    • Instruments must have been properly installed, calibrated and maintained according to the manufacturer’s recommendations
    • Clinical samples are potentially infectious and must be processed under a laminar flow hood/
    • The experiment must be performed according to good laboratory practices
    • Do not use this kit after expiration date
    • Many freezing/thawing cycles of the reagents must be avoided, and could lead to decrease of sensitivity
    • Once defrosted, spin down briefly the tubes before use
    • Spin down briefly enzymes tubes to pellet the viscous content at the bottom of the tubes.
    • Use of ice or cooling block is mandatory to prepare the mix and dispense reconstituted mix in wells.
    • Define 3 working areas: 1)Isolation of DNA/RNA, 2) Preparation of the reaction mix and 3) Amplification/detection of amplified products.
    • Pipettes, reagents and other materials must not cross each area
    • Specific caution is required to preserve the purity of the reagents and reaction mixtures. The use of gloves is mandatory. Appropriate methods of preparation of DNA should be used.
    • Always use filtered tips for micropipettes
    • Use specific lab coat and gloves in each working area
    • Do not pipette with mouth and do not eat, drink or smoke in the area
    • Avoid sprays

    VII.               Collection of samples, transport and storage

    • Collect samples in sterile tubes
    • The samples should be extracted immediately or frozen from -20°C to 80°C
    • Transport of clinical samples must obey local regulations for this type of infectious agents.

    VIII.               Procedure

    VIII.1   RNA/DNA extraction

    RNA/DNA extraction kits are available from ADNUCLEIS.

    For semen samples, the RNA extraction kit and RNA/DNA purification kit of ADNUCLEIS is recommended.

    The RNA IPC on the Vic channel added to the RT-PCR reaction ensures that a negative result is not due to the presence of high amount of RT-PCR inhibitors. In that case, 3 µl of RNA IPC is added per reaction.

    VIII.2   Q RT-PCR

    General comment: Positive control contains a high concentration of nucleic acids. Manipulations must be performed carefully to avoid contamination.

    It is necessary to test the positive control PRRS NA RNA (12 µl in final volume of 52 µl reaction), as well as a negative control (elution buffer, supplied separately). RNA IPC is added in each RT-PCR well (3µl) or in the reconstituted mix at the appropriate volume (3 µl * number of reaction prepared).

    Example of qualitative real time RT-PCR plate

     

      1 2 3 4 5 6 7 8 9 10 11 12
    A Positive control + IPC
    B IPC
    C Negative control
    D Sample 1 + IPC
    E Sample 2 + IPC
    F …
    G
    H

    Positive control: PRRS NA synthetic RNA

    IPC: synthetic RNA to control inhibition properties of the sample.

    VIII.3   Real time RT-PCR protocol

    The Reverse transcriptase reaction is performed before the PCR. The Taq N polymerase is added after the reverse transcriptase reaction is performed.

     

    • Homogenize gently the premix tube before starting and spin down brief the premix tubes, enzymes tubes and control tubes.
    • Prepare the reaction mix as follows by multiplying the number of N samples to test (including positive, IPC, negative controls). On average, prepare enough reagents for N+3 reactions minimum.
    Number of reactions (N) 1 N+3
    Premix 18 µl (N+3)x 18 µl
    RT N 1 µl (N+3)x 1 µl
    IPC 3 µl (N+3)x 3 µl
    Elution buffer 12 µl (N+3)x 12 µl
    Total volume of Reaction mix 34 µl (N+3)x 34 µl

     

    • Distribute 34 µl of the reaction mix with a micropipette and filtered sterile tips in each tube or well of a microplate for real time PCR.
    • Add 12 µl of sample of extracted nucleic acid or RNA positive control for the positive control or Elution buffer for the negative control.
    • Close immediately with an adhesive fillm to avoid all contamination
    • Centrifuge briefly to collect all the PCR reaction mix at the bottom of the tubes or plate.
    • Run the following Program on the qPCR instrument for the reverse transcription:

     

    program Temperature duration Cycle (s)
    Reverse transcription 42°C 30 min 1

     

    Once the reverse transcription is done, the Taq N is added into each well.

    Dilute the Taq N as follows by multiplying the number of N sample to test ( including positive, IPC, negative controls). On average, prepare enough reagents for N+3 reactions minimum.

    Number of reactions (N) 1 N+3
    Taq N 2 µl (N+3)x 2 µl
    Elution buffer 8 µl (N+3)x 8 µl
    Total volume of Reaction mix 10 µl (N+3)x 10 µl

     

    Distribute 10 µl of the diluted Taq N with a micropipette and filtered sterile tips in each tube or well of a microplate for real time PCR.

    • Run the following Program on the qPCR instrument for the PCR amplification:
    program Temperature duration Cycle (s) Fluorescence acquisition
    DNA denaturation 95°C 5 min 1
    Amplification 95°C 15 s 42
    60°C 40 s yes
    Cooling* 14.8°C 2 min 1

    *temperature of the cooling step can vary according to the qPCR instrument. Please contact ADNUCLEIS for further information.

    Note 1: set heating and cooling rate by default, up to 20°C/sec, or 100 %

    Note 2: For Applied Biosystems, select “none” in “Passive reference”.

    Note 3: n RotorGene, please calibrate the signal by clicking n “GAIN OPTIMIZATION”

    IX.               Validation of the experiment

    For the assay to be valid, the Ct values for the controls must be the following.

    Outside of these values, the experiment is not valid.

    Controls PRRS NA RNA control IPC Negative control
    FAM <30 >35
    VIC <37 >35

    X.               Data analysis and interpretation

    For clinical samples, the following results are possible:

    FAM signal (target) VIC signal (IPC) Presence of the target Test validity/comment
    <35 >37 positive
    <35 <37 positive
    Not Ct or >35 <37 negative valid
    Not Ct or >35 Not Ct or >37 inhibited Possible inhibition of RT-PCR. Dilute ½ or 1/10 the extract, otherwise extract once again the sample and retest bt RT-PCR

    XI.               Performances analysis

    XI.1        Studies of repeatability and reproducibility

    Studies of repeatability and reproducibility of the kit ADNUCLEIS PRRS NA virus have been achieved using a synthetic RNA by in vitro transcription on a plasmid containing a fragment of the PRRS NA virus.

    The tables below indicate the coefficient of variation (CV) depending on the concentration of the RNA

     

    Table of Repeatability (intra-experiment) and reproducibility (inter-experimental)

    copies/PCR Répétabilité intra expériementale Reproductibilité inter experimentale
    1.55E+08 1.55 2.43
    1.55E+07 0.17 1.92
    1.55E+06 0.35 0.75
    1.55E+05 0.38 0.31
    1.55E+04 0.50 2.52
    1.55E+03 0.64 1.31
    7.76E+02 1.97 2.28
    1.55E+02 2.28 4.40
    7.76E+01 0.09 0.82
     
    Moyenne CV 0.88 1.86

     

    XI.2        Performance analysis

    • Analytical sensitivity: 3.38×101 copies/PCR for the PRRS NA virus
    • Linearity for quantification: from 7.76×102 copies to 1.55×108 copies per PCR

    *average Ct are calculated with the three replicates

    kit Channel Efficacy coefficient of correlation R² Slope
    SDRP-NA FAM 126.04 % 0.9941 -2.83

    *average efficacy, R² and slope are calculated with the three replicates

     

     

    XII.               Biobliography

    • Brar M. S., Shi M., Murtaugh M. P., Leung F. C. (2015). Evolutionary diversification of type 2 porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 96, 1570–1580.
    • Dekkers J, Rowland RR, Lunney JK, Plastow G. Host genetics of response to porcine reproductive and respiratory syndrome in nursery pigs. Vet Microbiol. 2017 Mar 22
    • Gillespie, J., Opriessnig, T., Meng, X.J., Pelzer, K., Buechner-Maxwell, V., 2009. Porcine circovirus type 2 and porcine circovirus-associated disease. J. Vet. Int. Med. 23, 1151–1163.
    • Hansen, M.S., Pors, S.E., Jensen, H.E., Bille-Hansen, V., Bisgaard, M., Flachs, E.M., Nielsen, O.L., 2010. An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark. J. Comp. Pathol. 143, 120–131.
    • Holtkamp DJ, Kliebenstein JB, Neumann EJ, et al. Assessment of the economic impact of porcine reproductive and respiratory syndrome virus on United States pork producers. J Swine Health Prod. 2013;21(2):72-84.
    • Zimmerman JJ, Karriker L, Ramirez A, Schwartz K, Stevenson G. Diseases of Swine. Oxford: Wiley-Blackwell Publishing; 2012. pp. 461–462.

    XIII.               Waste disposal

    Dispose of waste according to DASRI regulations

    Real Time PCR as an alternative to cultural methods

    Real-time PCR is known to allow the isolation, identification and quantification of the DNA of bacteria, viruses, parasites, allergens and GMOs in a very short time (1 to 3 hours) in all areas: food safety, animal and / or human health.

    For a long time, this technic was of limited use due to the cost of the detection of some food hygiene related pathogens, the short shelf life of some products, the goal of Professor Michel Franck, CEO ADNucleis, is to expand the application of this PCR technic to the identification and quantification of traditional hygiene indicators (TVC, E.coli, coliforms, Pseudomonas spp, Listeria spp), and / or pathogens (Salmonella, Listeria monocytogenes, Cronobacter sakazakii, E. coli STEC O157H7, viruses , parasites …). This goal has now been achieved, thanks to a new innovative method, at equivalent prices to the prices of traditional cultural methods.

    This method, especially when checking for routine contaminants on a production line, can be done with or without a specialized robotic platform, also developed by ADNucleis.

    Thanks to its industrial partners, ADNucleis continues its goal to make real-time PCR an industry standard though the simplification and automation of the entire analytical process.

    PCR Kit Cronobacter spp (Enterobacter sakazakii)

    An AFNOR certified Cronobacter spp (Enterobacter sakazakii) Real Time PCR kit is available at ADNucleis.

    ADNucleis has positioned itself in Food quality and hygiene market for the fast testing of milk powder and infant formula with its Cronobacter spp (Enterobacter sakazakii) PCR kit.

    The harmful effect of this bacteria on at risk individuals is well known. In France, the numerous cases of food poisoning, in the 90s early 2000s, with an infant mortality rate, among those affected, of about 40 to 80% are proof of these harmful effects.

    ADNucleis is AFNOR certified for its diagnostic product, Cronobacter spp (Enterobacter sakazakii) NF VALIDATION 16140.

    Like all ADNucleis PCR kits, the analysis is fast (less than 24 hours, including pre-enrichment) and the cost is particularly competitive since ADNucleis has reduced its price to that of culture method namely 2€ per amplification kit (all real-time PCR platforms are compatible with this analysis)

    Operator time is very short and can be fully automated, which gives PCR an undeniable competitive advantage; it is estimated at less than 2 minutes per sample; the rest of the time is instrument time (55mn).

    Contact communication :

    Virginie Morel

    R&D communication

    v.morel@adnucleis.com

    Automatisation

    ADNucleis provides automation, for PCR sample prep, with features including the following:

    • DNA and RNA extraction and purification with silica microbeads or columns,
    • Plate preparation, for an input of 1 to 3 sample plates and an output of up to 3 PCR plates,
    • Shaking, cooling and heating steps.

    Thanks to its innovative algorithms and washing processes, ADNucleis platforms perform protocols with a decontamination step in order to avoid the change of tips. The process launches automatically when needed, the user-friendly software allows the operator to selects the specific(s) target(s) for which his sample is being prepared, and the robot does the rest.

    ADNucleis’ extraction and purification protocols are functional with all robotic pipetting platforms with all the benefits of robotics: repeatability of operations, quality and traceability.

    We provide the choice of 2 methods: the faster method with disposable tips (1 minute 30 per sample) or more economically advantageous, without disposable tips with our needle decontamination protocol (3 minutes per sample)

    This protocol is compatible with large throughput (up to 100 000 extractions per year per unit).

    ADNucleis provides the protocols for extraction and purification by silicon beads or columns, and for the preparation of PCR plates.

    Our easy to use, innovative algorithms, allows the operator to easily prepare up to 3 PCR plate from plates of extracted DNA or RNA, with a needle decontamination step with each column change.