EXOone Erysipelothrix rhusiopathiae


Available formats

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

Erysipelothrix rhusopathiae is ubiquitous in the environment and has been reported as both saprophyte and etiological cause of disease in a wide range of animals including fish, birds, mammals and human.
Moreover, E. rhusiopathiae is recognized as the etiological agent of swine erysipelas, a disease of significant economic consequence. Four main disease syndromes are reported in porcine infections: acute septicemia, subcutaneous disease (diamond skin), endocarditis and arthritis. Transmission might be mediated by the intake of contaminated food and water or the direct contact between ill animals. Subclinical infected swine should not be neglected due to their potential shedding of the bacteria through their feces, saliva and nasal mucus.
The identification of this bacterium by classical methods might be difficult due to its potential for multiple cellular morphologies and its variable Gram stain results. Therefore the use of an assay for molecular detection improves the sensitivity and specificity of the analysis.
  • 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 Erysipelothrix rhusiopathiae OneMIX qPCR kit Ref. No. ERUS

For synovial fluids, joint tissue, lymph node, liver, lung, spleen, cultures and vaccines; from Swine and Small ruminants

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

For synovial fluids, joint tissue, lymph node, liver, lung, spleen, cultures and vaccines; from Swine and Small ruminants

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

1 tube
(100 rxns)

ERUS 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
ERUS 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) Ten field strains of E. rhusopathiae isolated from goat (n=1), sheep (n=4) and swine (n=5) resulted positive.

b) In order to test the specificity, 25 related microorganisms including main related bacteria were analyzed and all of them resulted negative.

c) A collection of clinical samples (n=61) taken from different species such as goat (n=7), sheep (n=34) and swine (n=20) affected by arthritis (n=50) or septicemia (n=11) was analyzed by EXOone qPCR E. rhusopathiae. Results showed 18 positive cases what means an almost perfect concordance when compared with microbiological isolations results (kappa=0.85; SE=0.07). Three samples whose Cq values were higher than 34 did not obtain microbiological isolation.

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

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

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