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

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