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Protocol
FMDV PLA assay
Keywords: Foot and Mouth Disease Virus, Foot and Mouth Disease, FMVD, Antigen
Describes a Proximity ligation assay (PLA) for the detection of Foot and Mouth Disease Virus (FMDV).
The Proximity ligation assay (PLA) can be used for detection of different pathogens such as FMDV (Foot and Mouth Disease Virus).
The basis of the PLA is that pathogen specific-antibodies binding target proteins are coupled to non-sense oligonucleotide strands (proximity probes). These proximity probes can be joined by ligation when two or more such reagents are brought into proximity by binding to the same target molecule or target molecule complex.
The DNA ligation products are subsequently detected by PCR amplification using fluorogenic probes to detect the amplified product.
The assay shows a high sensitivity and specificity that is in line with RT PCR.
Required Materials
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Preparation of probes
- D-Biotin-N-hydroxysuccinimide ester
- PBS , pH 7,4
- Streptavidin conjugated oligonucleotides , 30 nMol/l
- BSA , 10 g/l
- polyA bulk nucleic acid , 16 mg/l
- D-biotin , 1 mmol/l
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Tris-buffer
- Tris-base
- HCl
- Milli-Q water
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Assay
- PBS , or other dilution factor of choice.
- PCR tubes
- KCl , 50 mM
- MgCl2 , 3.15 mM
- T4 DNA ligase , 0,4 Weiss units
- Detection Kit , (connector oligonucleotide) 400 nM
- ATP solution , 80 µM
- dNTP kit (2 mM) , 200 µM of each.
- PLA Probe plus , (primer) 100 nM
- PLA Probe minus , (primer) 100 nM
- TaqMan® MGB Probes , 100 nM
- Platinum Taq DNA polymerase + buffer , 1.5 U
Instructions
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Preparation of tris-buffer (LY9PV5)
Description of how to prepare the tris-buffer needed for the ligation and TaqMan mix.
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General information
Name Tris base, Tromethamine Molecular Weight 121.13504 g/mol Molecular Formula C4H11NO3 XLogP -2.7 CAS No. 77-86-1 pKa(25ºC) 8.08 -
Preparation
- Prepare 800 mL of base solution.
- Add HCl soln. according to target pH.
- Adjust the pH of the solution.
- Add H2O up to 1L.
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Acid
target pH volume amount 7.4 70 mL 12×0.07 = 0.84 mol 7.6 60 mL 12×0.06 = 0.72 mol 8.0 42 mL 12×0.042 = 0.50 mol HCl soln: 12M, 37% w/w, hazardous!
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Preparation of proximity probes
Biotinylation of antibody
1) D-biotin-N-hydroxysuccinimide ester (Roche Diagnostics Corp. Germany) is mixed with the antibody in a 10-fold molar excess and with a volume ratio of 1:10.
2) The solution is left for four hour incubation at room temperature and at constant agitation.
3) The biotinylated antibody is dialysed against Phosphate buffered saline (PBS, pH 7.4) and stored at -20C until use.
Construction of proximity probes
1) Streptavidin conjugated oligonucleotides 3’ and 5’ (Olink Bioscience, Uppsala Sweden) are mixed with the biotinylated antibody; 30nmol/l antibody is mixed with 30nmol/l oligonucleotide 3’ and 5’ respectively in a final volume of 5µl and left for one hour at room temperature.
2) The proximity probes are diluted to a final concentration of 1.2nmol/l in a probe dilution buffer and stored at +4C until use.
Probe dilution buffer
PBS, 10g/l Bovine serum albumin (BSA), 16mg/l sheared polyA bulk nucleic acid (Sigma), 1mmol/l D-biotin (Molecular Probes)
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Assay procedure
Detection of FMDV by homogenous phase PLA
1) Dilute the samples 1 in 5 in PBS (or any other dilution factor of choice).
2) Mix 1µl of diluted sample with 4µl of a solution containing both proximity probes (each diluted to a concentration of 24 pM in probe dilution buffer) in optical PCR tubes (Applied Biosystems, US) and incubate for 1 hr at 37°C.
3) Add 50µl of the ligation and TaqMan PCR mix and incubate for 5 min at room temperature.
Ligation and TaqMan mix:
- 50 mM KCl
- 10 mM Tris-HCl (pH 8.3)
- 3.15 mM MgCl2
- 0.4 Weiss units of T4 DNA ligase (Fermentas)
- 400 nM connector oligonucleotide (Olink Bioscience, Uppsala Sweden)
- 80 µM ATP
- 200 µM each of the deoxynucleoside triphosphates
- 100 nM primers forward and reverse (Olink Bioscience, Uppsala Sweden)
- 100 nM TaqMan® MBG probe (Applied Biosystems)
- 1.5U Platinum Taq DNA polymerase (Invitrogen)
4) Transfer the tubes to a real-time PCR instrument and use the following temperature profile: 95°C 2 min, 45 cycles: 95°C 15 sec and 60°C 2 min.
Figure FMVD -
Interpretation of results
The assay cut-off value is set to two standard deviations (S.D.) over the background signal (CT value). Samples with values below this threshold are considered to be negative while samples with values higher than the threshold are positive.
References
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Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus. ,
Author: Nordengrahn A, Gustafsdottir SM, Ebert K, Reid SM, King DP, Ferris NP, Brocchi E, Grazioli S, Landegren U, Merza M | Date: Mar 2008