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Measurement of bovine leukemia proviral DNA

Series: Ernestly measurement

Measurement of bovine leukemia proviral DNA

Although this is not known very widely, enzootic bovine leukosis which is a type of bovine leukosis caused by the bovine leukemia virus has been increasing in recent years. We examined if the DNA (proviral DNA) integrated by the virus can be measured using a mobile PCR device.

Sample

Butchered meat (beef) whole blood

Pretreatment

Blood stored in a frozen state (-80 degrees Celsius ) was centrifuged and rinsed with PBS twice before being used as a sample.

Reaction composition

  μL Remarks (final concentration)
Enzyme: KAPA 3G Plant (2.5 U/μL) 0.6 1.6U/reaction
Buffer (x2) 8 x1
F-Primer (100μM) ※1 0.1 1000nM
R-Primer (100μM) ※1 0.1 1000nM
Probe (100μM) ※1 0.05 600nM
Sample 1  
PCR grade water 6.2  

Reaction sample total 16μL

Program

Process Temperature Time  
Initial denaturation 95℃ 15sec  
Annealing/extension 60℃ 10sec 45cycle
Denaturation 95℃ 3.5sec 45cycle
Results

The test result clarified that bovine leukemia proviral DNA can be measured from whole blood stored in a frozen state via direct PCR.

*1 Changed from the following literature

Primer/probe

Made by Nihon Gene Research Laboratories Inc. 

Reference literature: Journal of Virological Methods 189 (2013) 258– 264 We sincerely thank Miyakonojo Meat Inspection Center in Miyazaki Prefecture that provided cooperation for measurements.