EXOone Paratuberculosis (multiplex)


Available formats

  • EXOone oneMIX Kit 50rxn
  • EXOone Plex Kit 100rxn

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis in several animal species, especially ruminant, with worldwide occurrence. Manifestations of the disease are weight loss, reduced milk production, infertility and, in later stages, emaciation and diarrhea.
MAP control is hampered due to ineffective diagnosis, particularly in subclinically infected animal population. Other diagnostic tests, such as ELISA and fecal culture, are being used commonly. Though fecal culture is considered the gold standard, the method is time-consuming (6–8 weeks). Moreover, serological tests in apparently healthy or subclinically infected animals frequently give rise to false negative results. PCR methods increases specificity and sensitivity of diagnosis, as well as it reduces the time required for diagnosis.
  • Fast&Easy. All the reagents already included in a single tube:
  • Primer/Probe (pathogen, EC).
  • Mastermix with main reagents: enzymes and buffer.
  • Just mix 15µl of the oneMIX and 5µl of your sample.
  • Endogenous Control (EC) to avoid false negative results and ensure that the entire process of your qPCR diagnosis has been correctly performed.
  • Multiparametric: Combine as many parameters as you need in the same run. Therefore, customize your own panels and detect several pathogens simultaneously. Obtain lots of information in less than 90 minutes.

This kit contains:

1 tube of oneMIX mixture that includes MasterMix, Primer/Probe, polymerase and buffer. (1 tube -> 50 reactions, 2 tubes -> 100 reactions).

1 tube of Molecular grade water.

1 tube of Positive Control (quantifying standard).

Quick Protocol: EXOone Paratuberculosis (multiplex) OneMIX qPCR kit Ref. No. XPTB

For faeces, rectal swabs, milk and tissues including intestine, ileocecal valve and lymph nodes, cultures and vaccines; from Cattle and Small ruminants

Reference Description Presentation
XPTB OneMIX tube Each tube contains buffer, enzyme, dNTPs, primers/probes for pathogen and endogenous control detection Two tubes (2x50 rxns)
XPTB POSITIVE CONTROL Lyophilized specific synthetic control* 1 tube
Water Molecular grade water 1 tube (1 ml)
* See the Quality Control Certificate for copy number.

Required material but not provided in the kit
• Nucleic acid sample • Powder-free vinyl gloves
• Micropipettes • Plate cicrocentrifuge (optional)
• Sterile tips with filter •Real-time PCR thermocycler + software
Reconstitution of positive control
1. Spin briefly the Positive Control tube and reconstitute with 250 µl of Water.
2. To facilitate resuspension keep the tube for 10 min. at room temperature.
3. Vortex gently the tube before use.
4. Store reconstituted Positive Control at -20ºC.
Procedure
1. Thaw the oneMIX tube you need regarding the number of required reactions. Non used oneMIX tube must be kept frozen.
2. Vortex the oneMIX tube briefly.
3. Dispense 15 µl of oneMIX into each well/tube used for the assay.
4. Add 5 µl of nucleic acid sample or Positive Control or negative control (water) in the corresponding wells/tubes.
5. Seal the wells/tubes with their corresponding film or caps.
7. Centrifuge the plate/tubes gently before inserting it into the thermocycler to prevent drops in the well pit walls.

Thermal Protocol


Data Collection (FAM) Data Collection (FAM) Endogenous control (HEX). If your thermocycler has not HEX channel, use VIC channel. In Applied Biosystem thermocyclers do not use ROX as passive reference


SAMPLE (FAM)
Positive if Cq ≤ 40
POSITIVE
CONTROL
(FAM)
NEGATIVE
CONTROL
(FAM)
SAMPLE ENDOG.
CONTROL (HEX)
ASSAY
RESULT
Positive Positive Negative Do not consider* Valid
Positive Positive Negative Positive Valid
Positive Positive Negative Negative** Not Valid
Positive/Negative Negative Negative Positive/Negative Not Valid
Positive/Negative Positive Positive Positive/Negative Not Valid
*Do not consider EC signal in Positive samples or Positive control.
** Evaluate purity and concentration of the sample. If needed, prepare ten-fold dilutions (10-1 to 10-2) of this sample and re-test in the qPCR assay. In case of persisting negative results in EC, a new nucleic acid extraction should be done.

Absolute quantification. Standard curve preparation.
- Reconstitute Positive Control with 250 µl molecular grade water.
- Make 5 ten-fold dilutions (10-1 to 10-5) with Buffer A or nuclease-free grade water.
- Amplify 2-3 replicates in the same thermocycler run.


These figures should be considered only as an example of a Standard curve. Cq values might not match exactly with those obtained with this assay.

Revision No. 2. Issue Date: Junio, 2015

The simplest format that fulfill your needs.

  • Designed just for pathogen detection
  • Lyophilized reagents allow a room temperature shipment and makes the delivery easier.
  • Multiparametric: Combine as many parameters as you need in the same run. Therefore, customize your own panels and detect several pathogens simultaneously. Obtain lots of information in less than 90 minutes.

This kit contains:

1 tube of 100 reactions.

1 tube of reconstitution buffer (Buffer A).

1 tube of Positive Control (quantifying standard).

Quick Protocol: EXOone Paratuberculosis (multiplex) Basic qPCR kit Ref. No. XPTB_BAS

For faeces, rectal swabs, milk and tissues including intestine, ileocecal valve and lymph nodes, cultures and vaccines; from Cattle and Small ruminants

Reference Description Presentation
XPTB Basic qPCR tube Tube contains lyophilized primers and probe for specific pathogen detection

1 tube
(100 rxns)

XPTB POSITIVE CONTROL Lyophilized specific synthetic DNA* 1 tube
Buffer A Rconstitution Buffer / Negative control 1 tube
(1.5 ml)
* See the Quality Control Certificate for copy number.
NOTE: For a best performance of the assay store reagents at -20ºC. after arrival.

Required material but not provided in the kit
• Nucleic acid sample • Powder-free vinyl gloves
• Micropipettes and sterile tips with filter • Microcentrifuge (optional)
• Real-time PCR thermocycler + software • qPCR / RT-qPCR Master Mix
Reconstitution of lyophilized reagents
Reconstitute Basic qPCR tube and Positive control separately.
1. Spin briefly (15 sec) to ensure the material is in the bottom of the tube.
2. Reconstitute Basic qPCR tube (100 μl) and Positive Control (250 μl) with Reagent A. Keep the tube for 10 min. at room temperature.
3. Vortex gently before use.
4. Store reconstituted tube at -20ºC.
Preparation of real-time PCR assay
The qPCR setup will depend of your reagents concentration, please use the following pippeting squeme as a model:

Stage Component Per Sample
Preparation of Master Mix 2X qPCR Master mix Buffer
XPTB Basic qPCR tube mix
Nuclease-free water
10 μl
1 μl
up to 15μl
Total Volume 15 μl

Preparation of

qPCR assay

Master Mix
Sample*
15 μl
5 μl
Total Volume 20 μl

*Make sure that at less one negative control (water) as well as one positive control are included in the PCR run.


Thermal Protocol
(FAM) In Applied Biosystem thermocyclers do not use ROX as passive reference

Interpretation of Results:
A sample is considered positive if Cq value is ≤ 40. Positive control should result positive with a Cq value ranging those stablished in the QC sheet of this kit. No amplification should be observed for the Negative control. For a detailed interpretation of the results please refer to the User's manual of this kit.

Absolute quantification. Standard curve preparation.
- Reconstitute Positive Control with 250 µl of Buffer A.
- Make 5 ten-fold dilutions (10-1 to 10-5) with Buffer A or nuclease-free grade water.
- Amplify 2-3 replicates in the same thermocycler run.



These figures should be considered only as an example of a Standard curve. Cq values might not match exactly with those obtained with this assay.

Revision No. 2. Issue Date: Junio, 2015

a) A total of 76 clinical cases including 30 positive cases were compared with this assay and other commercial qPCR kit. The agreement was almost perfect (Kappa 0.89) between the two kits. Furthermore, 12 of these clinical cases were confirmed by positive immunoperoxidase staining or IFAT method using mAb anti-Mycobacterium tuberculosis clone 1038/103.
b) A specificity panel of 36 related microorganisms including: other species of Mycobacterium (2), bacteria (31) and virus (3), resulted negative.
c) A panel of 114 clinical cases including: 56 faeces, 38 digestive tissues, 16 rectal swabs and 4 milk was evaluated with this qPCR assay. MAP was identified in 29.8% of these cases.
d) This kit was also validated through a collaboration project with INVESAGA research group (Lugo, Spain). 81 microbiological isolations gently provided by INVESAGA resulted positive. Besides, a collection of 69 samples of feces previously tested by our collaborator was analyzed with EXOone Paratuberculosis. That panel included 33 positive samples and 36 negative specimens already tested by other commercial qPCR resulting an almost perfect concordance (kappa value = 0.94; SD = 0.04)

• We studied the reportable range for this qPCR assay using a purified specific synthetic oligonucleotide (PTBC positive control).

• This qPCR kit can identify MAP from clinical samples and vaccine/field strains.

How to interpret the results:
IS900 pos and F57 pos confirmed as MAP
IS900 pos (Cq<33) and F57 neg confirmed as not MAP
IS900 pos (Cq>33) and F57 neg not confirmed as MAP