Clinical significance
JD is prevalent worldwide and imposes considerable economic burden to the cattle industry due to production losses. Clinical manifestation of JD appears too late, more than a year after infection, making difficult an early diagnosis of the disease, thus, only PCR can provide early diagnosis and eradication of infection from the herd.
Principle of analysis
The method is based on the isolation of total DNA from samples, and further amplification of specific regions of MAP DNA (IS900 gene) using specific oligonucleotide primers and the synthesis of complementary DNA chains using the enzyme Hot start Taq-DNA polymerase. Fluorescent detection of the analysis results is performed both in real-time (Real-time mode) or end point assay using two fluorescence detection channels (FAM525 nm, ROX580 nm), see table below:
| Marker |
Detection channel |
| MAP, IS900 gene |
FAM |
| Internal control, 16S gene |
ROX |
Kit contents
| Component name |
Quantity |
| MAP Master Mix (Lyophilized) |
1 vial |
| MAP Positive Control (Lyophilized) |
1 tube |
Storage and stability
The test kit reagents are stable for the shelf life indicated on the label (at least 12 months from manufacture date) if stored at room temperature.
Sample preparation
Tissues, swabs, feces and cultural material samples can be processed according commercially available DNA extraction kit recommendations. We recommend using our Nucleic acids extraction kit compatible with automatic Nucleic extraction device LTEXT1.
Reagent preparation
Master Mix preparation: Add distilled water 0,5 mL directly to Lyophilized Master Mix, wait 5 min, mixing well and dispense 5 µL into PCR testing tubes.
Positive control preparation: Add distilled water 0,5 mL directly to Lyophilized Positive Control, wait 5 min, mixing well and use directly 5 µL for PCR or use 100 µL for Nucleic acid extraction (to control Nucleic acid extraction efficiency). The rest of TE buffer can be used as Negative Control.
Reagents and controls must be kept in a chilled stand. After use reagents and controls can be freeze at minus 20°C for storage. Several freezing-thawing cycles are allowed.
Assay procedure
1. Select the required amount of the PCR tubes or wells in the PCR plate.
2. Using pipette tips with an aerosol filter dispense Master mix 5 µL add 5 µL of DNA samples (Positive and Negative control samples).
3. Place testing tubes in the fluorescence PCR instrument, and set the negative/positive control and unknown samples parameters for PCR reaction according to the instrument operating instructions. Fluorescence is measured at 60°C on the FAM, and ROX channels. The PCR parameters are shown in table below:
| Step |
Temp |
Time |
Repeats |
Data collections |
| 1 |
37 C |
10 min |
1 |
No |
| 2 |
95 C |
5 min |
1 |
No |
| 3 |
95 C |
10 sec |
45 |
No |
| 3 |
60 C |
30 sec |
45 |
Yes |
4. Start run.
Result records and their interpretation is shown in the table below:
| Marker |
Fluorescent channels |
Pasteurella Positive |
Pasteurella Negative |
| MAP IS900 gene |
FAM |
Ct≤ 45 |
Undetectable |
| Internal control gene 16S |
ROX |
Ct≤ 35 |
Ct≤ 35 |
