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Wild-type Strain and gE-deleted Vaccine Strain of Pseudorabies Virus Detection Kit

Wild-type Strain and gE-deleted Vaccine Strain of Pseudorabies Virus Detection Kit

Wild-type Strain and gE-deleted Vaccine Strain of Pseudorabies Virus Detection Kit enables detection of the PRV-HE in swine blood, serum, or tissues by real‑time PCR.

For Veterinary Use Only. For In Vitro Use Only.

Specification

Precautions

Storage

Validity Period

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Product Information

WARNING! Follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Product description

The Kit enables detection of the PRV-HE in swine blood, serum, or tissues by real‑time PCR.

The assay is a single‑well real‑time PCR in which PRV-HE and Internal Control (IC) targets are amplified and detected using fluorescent TaqMan® probes.

The kit includes:

  • PRV-HE-Detection solution: Contains primers, TaqMan® probes, dNTP, Mg2+, buffer, IC, etc.
  • PRV-HE-Enzyme mix: Contains Reverse transcriptase and DNA polymerase.
  • PRV-HE-Reverse transcriptase: Reverse transcriptase system.
  • PRV-HE-Positive control: Cloning plasmid containing target gene of PRV-HE.
  • PRV-HE-Negative control: Sterilized normal saline

Required materials not supplied

Unless otherwise indicated, all materials are available through other major laboratory supplier.

Item
Real-Time PCR System
96-well plate, strip tubes (8- or 12-wells), microtubes or capillaries compatible with thermal cycler used
Nuclease-free pipettes and filtered pipette tips
Two ice buckets or refrigerated racks:
• One for the PCR setup area where the PCR master mix is prepared
• One for the area where DNA samples and controls are prepared
Plate covers or caps compatible with the plates, strip tubes, microtubes, or capillaries
Nuclease-free reagent tubes for preparing master mix
Nuclease-free Water (not DEPC-Treated)
1× TE Buffer

Procedural guidelines

For each real‑time PCR run, include the controls indicated in "Set up the PCR reactions".

Requirements for input DNA

We recommend using the BIOPHARMAVET™  Pathogen DNA/RNA Kit for DNA extraction from biological samples, but you can also use other high quality DNA extraction methods after proper validation in your laboratory. In addition, prepare mock-purified sample using nuclease‑free water as the starting material and the same DNA isolation method used for test samples.

Before you begin

  1. Thaw reagents and samples:
    1. Thaw reagents and controls in an ice bucket or refrigerated rack.
    2. Thaw DNA samples in a separate ice bucket or refrigerated rack.
  2. Thoroughly mix the contents of each tube by vortexing, then briefly centrifuge.
  3. Store thawed reagents, controls, and samples at 2–8°C until use.

Set up the PCR reactions

  1. Add reagents, sample or control to the appropriate number of PCR plate wells, strip tubes, or capillaries according to the following table:
Sample type Volume per reaction
PRV-HE-Detection solution 10µL
PRV-HE-Enzyme mix 10µL
Test sample/Positive control/Negative control 5.0 µL

       2. Seal each plate or tube, mix, then centrifuge briefly to bring the contents to the bottom of the plate wells or tubes.

Guidelines for data analysis

  1. Following the manufacturer's instructions, set up the real‑time PCR run using the following
  • Reaction volume: 25 µL
  • Select detectors and assign TaqMan® probe reporter dyes for each well, tube, or capillary used in the analysis.
Target Reporter
PRV-gH FAM™  dye
PRV-gE HEX™  dye
IC ROX™  dye
  • Thermal cycling program:

Take BIOERTM FQA-48a as an example:

Stage Repetitions Temperature Time
1 1 50°C 2 minutes
2 1 95°C 5 minutes
3 40 95°C 15 seconds
60°C 30 seconds*
4 1 25°C 10 seconds

*Run the Thermal cycler program, collecting real‑time amplification data during stage 3. 

Validation criteria

The test is validated if the following criteria are met:

Reaction type PRV-gH PRV-gE IC
(FAM™ dye) (HEX™ dye) (ROX™ dye)
Positive control Ct ≤36 Ct ≤36 Ct ≤36
Negative control NO CT NO CT Ct ≤36

Interpretation of results

PRV-gH PRV-gE IC Interpretation
(FAM™ dye) (HEX™ dye) (ROX™ dye)
Ct ≤40 Ct ≤40 Ct ≤36 PRV-gE is detected.
Ct ≤40 NO CT Ct ≤36 PRV-gE-deleted is detected.
NO CT NO CT Ct ≤36 PRV is not detected.
/ / Ct ≤36 Invalid result.

Retest samples with invalid results

  1. Dilute the DNA samples 1:5 in 1X TE buffer.
  2. Repeat the real‑time PCR procedure with 5 µL of the diluted DNA, then interpret the results as follows.
  3. For diluted samples with invalid results, repeat the DNA isolation procedure on a new aliquot of the original sample lysate, then repeat the test.

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