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MycXtra® Fungal DNA Extraction Kit
What is the MycXtra® kit used for?
The MycXtra® Fungal DNA Extraction Kit is designed for the isolation and purification of fungal DNA present in human bronchoalveolar lavage (BAL), sputum and other lower respiratory tract samples from patients with suspected invasive fungal infection. Fungal DNA extraction is a critical first step to analyzing a sample for fungal specific DNA.
Is the kit CE marked?
Yes. Further performance evaluations with clinical samples are also underway. The kit has been initially tested for extraction of Aspergillus spp. and Pneumocystis jirovecii. Other fungi will also be evaluated.
How does the kit work?
As fungal cell walls are difficult to break down, a combination of processes are required to optimise yield; fungal spores and hyphae present in the sample are concentrated by centrifugation and re-suspended in a small volume of liquid. The sample is then added to a tube containing beads, lysis solution, bead solution and Inhibitor Removal Solution (IRS). The principle is to lyse the microorganisms in the sample using a combination of detergent and mechanical force using a vortex. The released DNA is bound to a silica spin filter; the filter is washed, and the DNA is eluted. The resulting DNA is of high purity and suitable for Real-Time PCR analysis and other downstream PCR applications.
How long does the procedure take?
Using BAL as a clinical sample, the extraction procedure takes between 45 minutes and one hour. If a viscous sputum sample is used, a pre-treatment step means the full extraction procedure may take between one and 1.5 hours.
What is included in the kit?
The kit is sufficient for 10 patient samples:
| Description | Volume (mL) |
|---|---|
| 10 x 2 mL Bead Solution tubes | 0.55 |
| Solution S1 | 0.65 |
| IRS* solution | 2.2 |
| Solution S2 | 2.75 |
| Solution S3 | 14.5 |
| Solution S4 | 3.3 |
| Solution S5 | 1.1 |
*Inhibitor Removal Solution
- 10 x Spin filters units in 2 mL tubes
- 40 x Microcentrifuge tubes (2 mL)
- Instructions for Use
Is OK to store respiratory samples at -20oC before extraction (instead of -80oC)?
Yes, though there may be slight loss of DNA signal over a prolonged period of storage at -20oC
What is the optimum volume of BAL to use with the MycXtra® kit?
As a guide, we suggest 2.0 mL minimum. The starting volume, and whether the sample was centrifuged prior to sampling, both affect sensitivity. The sensitivity of subsequent Real-Time PCR assays will decrease with lower volumes of starting material and may increase with higher volumes; yield is actually related to fungal load (how many spores/hyphae are present in the respiratory sample) i.e. how heavily colonised/infected the patient is.
What is the optimum volume of sputum to use with the MycXtra® kit?
Please use the maximum volume available.
Are any pre-treatments of viscous BAL/sputa required?
Yes, It is recommended that the BD BBLTM MycoPrepTM Specimen Digestion/ Decontamination Kit for Processing of Mycobacterial Specimens (Cat. No.240862) is used with the MycXtra ® protocol for the preparation of viscous respiratory specimens.
An alternative process is in development at Myconostica.
Can the MycXtra® kit be used with blood samples?
No, but a kit for the DNA extraction of fungal pathogens from blood is in development.
What additional equipment is required for use with the MycXtra® kit?
- Micro centrifuge (10,000 x g)
- General purpose centrifuge
- Micro-pipettes with filter tips
- Vortex Genie ® 2 (available from local suppliers)
- Vortex Plate Adaptor (available from Myconostica REF 080-015)
Is the MycXtra® kit fungal-DNA free?
Using standard PCR technology, the kit is free from detectable fungal contamination. It produces no detectable signal for Aspergillus spp. or Pneumocystis jirovecii in Real-Time PCR tests. It is essential that dedicated pipettes are used and not those used for setting up cultures or other fungal processing.
How is the MycXtra® kit shipped and stored?
It is shipped at ambient temperature and should be stored between 15oC and 30oC until the expiry date indicated on the kit box label, at which time it should be disposed of according to local regulations. Do not freeze. Do not open until ready to use.
How long is the expiry/shelf-life of the MycXtra® kit?
18 months
Does the MycXtra® kit have a positive control?
No, there is no positive control in the MycXtra® kit. The principal reason for this is that it is almost impossible to provide a suitable one. If we supplied the following, for example:
- A DNA sample would not reflect the extraction process or its efficiency
- Live Pneumocystis propagules in solution could be dangerous material for a mycology lab and may lead to the possibility of contamination.
- Attenuated or killed pathogens may not behave in the same way as extracting DNA from live pathogens with the kit
We would suggest that if a user wishes to investigate this further, they consider culturing Aspergillus AF293, possibly obtained from a fungal culture collection, dilute the culture to a known concentration and test spores as a positive control with the MycXtra® kit.
N.B. Every lot/batch of MycXtra® manufactured by Myconostica is QC tested to check that its components extract fungal DNA from spores.
How do I test extraction reagents for Aspergillus fungal contamination?
All reagents in the extraction process should be free from Aspergillus DNA contamination to prevent false positive results. If you observe Aspergillus contamination in negative extraction control samples and suspect contamination of extraction components, Myconostica recommend testing for Aspergillus contamination. Please see ‘TN3: Potential Aspergillus fungal contamination of reagents used in the DNA extraction process’ (TN3) in the Technical Note section for a detailed description of contamination testing methods.
For any further questions, please contact [email protected]