Detection of Fish Pathogens by Loop-Mediated Isothermal Amplification (LAMP) Technique


Target virus

Primer sequences (5′–3′)

References

VHSa

F3—GGS AAG CAA GGA YCA CGA G

[13]
 
B3—CAG GTG TCC YTC TAG TGT TTC
  
FIP—GAT CCA CCG ATA CTG TTT TTG GGG TTT TCC CGT TCT TCC CTG AAC CC
  
BIP—ARG GGG TYT GCA CAR CCT CGC TTT TCG ACK YGG GRC AAK GGG C
  
FLP—GTT ATG TCC TTA TGG ACA TTG
  
BLP—GTC AAA CTC ATT GGC AGG G
 
IPNVb

F3—CCA ATC TGC GGT GTA GAC AT

[14]
 
B3—CAT CAG CTC TCC CAG GTA CT
  
FIP—CCT CCT CGT CCA CTC CTG GTT TTT CCA TCG CAG CCC ATG AAC
  
BIP—TGC GAA ACA CAT CCC TGG CCT TTT TCT TGT TGG AGC CCT TTG C
  
FLP—CGA TGA GTG GCA GCC CTT
  
BLP—GAT CCA GAC CGG AAC CCT G
 
CyHV-3c

F3—TGC AGC AGC CCT TCA AG

[16, 18]
 
B3—GAC ACA CCG CCT GGT AAG
  
FIP—TGC ACA CCG CCG TCA GCT CAG GTG ACG GCG TTG GT
  
BIP—GAA GTG CAA GAT GCG CGA CGA CTC GGC GCC TCC AA
  
FLP—GTC CAG CTT GTC CGC CAT G
  
BLP—CAC CCT TCA CCG TCA GAA TCT C
 

aViral hemorrhagic septicemia virus

bInfectious pancreatic necrosis virus

cCyprinid herpesvirus-3




 


2.

Primers stock solutions: primers are ordered and come from the synthesis company with a specification sheet that usually contains all the information required to rehydrate the primers. For each of the LAMP primers, add the recommended amount of PCR-grade water and mix it well to get a primer stock solution of 100 pmol/μl. Prepare several aliquots from these stocks to avoid degradation by repeated freezing and thawing, and keep it at −20 °C until used.

 

3.

Primers working solutions: the working concentrations of LAMP primers solutions may be as following—10 pmol/μl of F3 and B3; 80 pmol/μl of FIP and BIP; and 40 pmol/μl of FLP and BLP. Prepare 100 μl of each LAMP primer working solution as indicated below.



  • Primers F3 and B3: mix 10 μl from the correspondent 100 pmol/μl primer stock solution with 90 μl of PCR-grade water.


  • Primers FIP and BIP: mix 80 μl from the correspondent 100 pmol/μl primer stock solution with 20 μl of PCR-grade water.


  • Primers FLP and BLP: mix 40 μl from the correspondent 100 pmol/μl primer stock solution with 60 μl of PCR-grade water.


  • Primer mix: prepare the primer mix by thorough mixing of equal volumes from each LAMP primer working solution in a new microcentrifuge tube, and keep it at −20 °C until used (see Note 2 ).

 





2.2 Reagents and Other Materials for LAMP Reactions and Direct Product Detection




1.

10× LAMP reaction buffer: 20 mM Tris–HCl, pH 8.8, 10 mM KCl, 1.5 mM MgSO4, 10 mM (NH4)2SO4, 0.1 % Triton X-100 (e.g., 10× Thermopol® reaction buffer, New England BioLabs, GmbH, Frankfurt, Germany).

 

2.

5 M betaine solution.

 

3.

100 mM dNTPs mix solution.

 

4.

25 mM MgSO4 (see Note 3 ).

 

5.

Bst DNA polymerase, large fragment (New England BioLabs, GmbH, Frankfurt, Germany).

 

6.

PCR-grade water.

 

7.

Enhanced Avian Myeloblastosis Reverse Transcriptase (eAMV) (Sigma–Aldrich, GmbH, Schnelldorf, Germany).

 

8.

SYBR Green I nucleic acid stain, 10,000× concentrate in DMSO. To prepare the working dilution (1:10 in DMSO), mix 1 μl of SYBR Green I nucleic acid stain 10,000× concentrate with 9 μl DMSO, and then make aliquots of 1 μl each and keep it at −20 °C until used.

 

9.

Fluorescent detection reagent (FDR) (Eiken Chemical Co. Ltd., Japan).

 


2.3 Detection of LAMP Products Using Lateral Flow Strips




1.

Lateral flow strips for nucleic acid detection (Milenia GenLine HybriDetect, Milenia Biotec GmbH, Bad Nauheim, Germany). It contains dipsticks and assay buffer.

 

2.

5′ end biotin-labeled FIP primer (prepared as in Subheading 2.1).

 

3.

5′ end FITC-labeled DNA probe (see Note 4 ).

 



3 Methods


LAMP reaction mixtures should be prepared on ice. After thawing, LAMP reagents and primers should be thoroughly mixed by vortexing and then spin-down and kept on ice until used. Carefully follow all waste disposal regulations when disposing waste materials.


3.1 Amplification of DNA Templates by LAMP




1.

LAMP reactions are usually carried out in a total volume of 25 μl.

 

2.

Prepare the master mix, which consists of all the reagents needed for one sample multiplied by the number of samples to test plus the positive and no-template controls and one additional sample to overcome eventual pipetting errors.

 

3.

The reagents needed for one LAMP reaction are 2.5 μl of 10× reaction buffer, 8 μl of 5 M betaine solution, 0.7 μl of 100 mM dNTPs solution (see Note 5 ), 3 μl of primer mix (see Note 6 ), 2 μl of 25 mM MgSO4 solution (see Note 3 ), 1 μl (8 units) of Bst DNA polymerase, and 5.8 μl PCR-grade water to complete the volume to 23 μl.

 

4.

After mixing the reagents of the master mix, dispense 23 μl in each tube labeled with the sample number.

 

5.

Add 2 μl of sample DNA to each tube (see Note 7 ). In the positive control tube, add 2 μl of the relevant DNA solution, while in the no-template control, add 2 μl of PCR-grade water.

 

6.

Incubate the reaction tubes in a water bath or heating block at 65 °C for 1 h.

 

7.

After incubation, inactivate the enzyme at 85 °C for 3 min and proceed to the products detection methods.

 


3.2 Amplification of RNA Template by RT-LAMP




1.

Perform as described previously for the amplification of DNA templates (in Subheading 3.1) with the following modifications: the reaction mixture will contain 2.5 μl of 10× reaction buffer, 8 μl of 5 M betaine solution, 0.7 μl of 100 mM dNTPs solution (see Note 5 ), 3 μl of primer mix (see Note 6 ), 2 μl of 25 mM MgSO4 solution (see Note 3 ), 1 μl (20 units) of eAMV reverse transcriptase (see Note 8 ), 1 μl (8 units) of Bst DNA polymerase, and 4.8 μl PCR-grade water to complete the volume to 23 μl.

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Mar 17, 2017 | Posted by in GENERAL | Comments Off on Detection of Fish Pathogens by Loop-Mediated Isothermal Amplification (LAMP) Technique
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