EXOone Encephalitozoon cuniculi


Available formats

  • EXOone oneMIX Kit 50rxn
  • EXOone oneMIX Kit 100rxn
  • EXOone Basic Kit 100rxn

Encephalitozoon cuniculi is an obligate intracellular parasite belonging to the phylum Microsporidia; It can infect mammals including humans in which is an important opportunist agent, especially within immunocompromised specimens, causing diarrhea, rhinosinusitis, keratoconjunctivitis, nephritis or hepatitis. The main host is rabbit, and infections can course subclinically. Nervous system, kidney and eye are the preferred targets of E. cuniculi.
E. cuniculi spores can be excreted in high quantities by urine. These spores are very resistant in environment and can infect water, food and other sources that could reach human consume.
Conventional methods such as serology and microscopy are reported as unreliable for diagnosis of encephalitozoonosis in domestic rabbits. qPCR technique appears as a opportunity to apply a sensitive and specific tool in the detection and identification of this parasite.
  • 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 Encephalitozoon cuniculi OneMIX qPCR kit Ref. No. ECUN

For kidney, semen and urine, cultures and vaccines; from Rabbit

Reference Description Presentation
ECUN OneMIX tube Each tube contains buffer, enzyme, dNTPs, primers/probes for pathogen and endogenous control detection Two tubes (2x50 rxns)
ECUN 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 Encephalitozoon cuniculi Basic qPCR kit Ref. No. ECUN_BAS

For kidney, semen and urine, cultures and vaccines; from Rabbit

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

1 tube
(100 rxns)

ECUN 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
ECUN 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) Sixteen field isolates of E. cuniculi resulted positive.

b) A panel of specificity consisting of 36 related microorganisms including pathogenic bacteria, virus, parasites and biological contaminants of the environment resulted negative.

c) A collection of 152 clinical cases, mainly from kidney tissue (n=105), semen (n=29), and other samples (n=18) was tested with EXOone E. cuniculi. Some of them (n=54) were compared with other commercial qPCR kit which detects Encephalitozoon spp., resulting a very good correspondence (Kappa=0.92).

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

• This qPCR kit can identify E. cuniculi from clinical samples and field strains.

• Maximum quantification limit of E. cuniculi ≥1010 copies/rxn.
• Minimum quantification limit of E. cuniculi = 50 copies /rxn.