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Protocol
One-step RT-LAMP assay
Keywords: RT-LAMP, RT, LAMP, Loop-mediated isothermal amplification
This RT-LAMP assay provides a number of benefits for the diagnosis of SVD. Since the assay is sensitive and rapid, and the isothermal amplification strategy used is not reliant upon expensive equipment it is particularly suited for “front line” diagnosis of SVD in modestly equipped laboratories, in field stations or in mobile diagnostic units.
Loop-mediated isothermal amplification (LAMP) is a method used to amplify RNA and DNA. Two or three primer pairs are used to amplify the template which gives a long stem- loop product under isothermal conditions. This report describes a one-step reverse transcriptase (RT) LAMP assay for the sensitive (50 viral RNA copies per assay) and rapid detection (within 30–60 min) of swine vesicular disease virus (SVDV). The results can be visualized either by gel electrophoresis or by the naked eye through the addition of SYBRGreen.
Instructions
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General aspects:
The highly conserved 3D polymerase gene was chosen as target for the primer design. The RT-LAMP reaction is carried out in a 25 µl mixture. After production of the master mix, 23 µl portions are filled into each reaction tube and 2 µl template are added.
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Master mix preparation:
The volume of the master mix depends on the number of extracted samples plus the number of positive and negative controls (water). Recipe and a template to calculate final volume can be found in the adjoined protocol.
Before dividing up the master mix into portions of 23 µl per tube, the master mix must be homogenised thoroughly.
The RT-LAMP reaction is carried out in a 25 µl mixture containing 1 × Thermo buffer (New England Biolabs, Beverly, MA, USA), 0.54 × First strand buffer (Invitrogen, Carlsbad, CA, USA), 1.12 mM dNTPs (GE Healthcare, Uppsala, Sweden), 0.2 µM each of F3 and B3, 1.6 µM each of FIP and BIP, 0.8 µM each of Floop and Bloop, 0.8 µM betaine (Sigma), 4.5U cloned AMV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 8U Bst DNA polymerase, Large Fragment (New England Biolabs).
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Addition of template RNA:
2µl template RNA is added to each tube with 23µl master mix, except for the negative control for which water is added. After addition of template RNA, close the reaction tubes and run LAMP.
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Thermodynamic profile:
Reverse Transcription/incubation 63°C 60 min Inactivation 80°C 2 min -
Analysis of results:
Run 5µl of the RT-LAMP product on a 1.5% agarose gel.
Digest 2µl product with BspHI (New England Biolabs) for 1 h at 37°C before analysis by gel-electrophoresis to confirm the result.
Add 0.5µl 10× Sybr-Green Gel Stain I 10,000× concentration (Molecular Probes, Leiden, the Netherlands) to 10µl RT-LAMP product to visualised the result. After a short vortex a colour change can be seen if the sample is positive.
References
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A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus ,
Author: Anne-Lie Blomström, Mikhayil Hakhverdyan, Scott M. Reid, Juliet P. Dukes, Donald P. King, Sándor Belák, Mikael Berg | Date: January 2008