Real-Time Reverse Transcriptase PCR for the Detection of Bluetongue Virus


Oligo name

Sequence (5′–3′)

Working concentration (μM)

Primer Hofm_BTV_IVI_F2

TGG AYA AAG CRA TGT CAA A

20

Primer Hofm_BTV_IVI_R2

ACR TCA TCA CGA AAC GCT TC

20

Probe Hofm_BTV_IVI_Pa

FAM-ARG CTG CAT TCG CAT CGT ACG C-Tamra

5


aProbe labeled with FAM (carboxyfluorescein) fluorophore and Tamra quencher




 


8.

Aerosol-resistant pipette tips.

 

9.

Adhesive PCR film.

 

10.

Marker pens (waterproof) and adhesive labels.

 





2.2 Equipment




1.

PCR cabinet.

 

2.

Calibrated pipettes.

 

3.

Freezer (−5 to −30 °C).

 

4.

Refrigerator (1–8 °C).

 

5.

Ice bucket with ice.

 

6.

Large capacity centrifuge.

 

7.

Real-time PCR machine.

 



3 Methods



3.1 Sample Preparation




1.

dsRNA from BTV test samples should be prepared as per standard methods (e.g., Qiagen/Roche viral nucleic acid extraction kits) and stored at −50 to −90 °C. BTV negative and positive control samples of known origin should also be prepared (see Note 2 ).

 


3.2 Assay Setup




1.

In a PCR clean room, prepare the one-step real-time RT-PCR reaction master mix according to Table 2. Sufficient volume of the master mix should be prepared to allow for testing of the required number of samples (see Note 3 ).


Table 2
Composition of the master mix for one-step real-time RT-PCR





































Master mix composition

Volume for 1 reaction (μl)

2× Superscript III RT-PCR reaction mix

12.5

Primer Hofm_BTV_IVI_F2 (20 μM)a

0.5

Primer Hofm_BTV_IVI_R2 (20 μM)a

0.5

Probe Hofm_BTV_IVI_P (5 μM)a

1.0

RNase-free water

2.5

2.5 μM ROX l

0.5

50 mM MgSO4

1.0

Superscript III RT/Platinum Taq

0.5

Total volume

19


aThe final concentration of the primers and probe per reaction is 0.4 μM and 0.2 μM, respectively

 

2.

Working in a clean laminar flow cabinet (see Note 4 ), add all reagents to a suitable container (e.g., microcentrifuge tube) allowing for the total volume of reagents (see Note 5 ). Mix the reagents gently with a pipette. Once outside the PCR clean room, maintain the master mix on ice and shielded from light.

 

Mar 17, 2017 | Posted by in GENERAL | Comments Off on Real-Time Reverse Transcriptase PCR for the Detection of Bluetongue Virus
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