FAQ LOOXSTER® Sample Preparation Kit

1. What is LOOXSTER®?

LOOXSTER® is a sample preparation kit especially designed for the separation, isolation and concentration of bacterial and fungal genomic DNA from total genomic DNA preparations obtained from a variety of samples.

Principle

2. What is the principle of LOOXSTER®?

Whole blood samples of sepsis patients contain complex DNA mixtures with a high human DNA background causing a very low bacterial plus fungal/human DNA ratio (1:2 x109). At first, a total cell lysis protocol is performed. The following concentration step uses the PUREPROVE® affinity chromatography incorporated in LOOXSTER®. PUREPROVE® is based on a DNA binding protein which recognizes definite unmethylated CpG motifs (A/T[CpG]A/T). Those motifs are predominantly present in bacterial and fungal genomes with different frequencies:

  • Bacteria:  1:50
  • Fungi:       1:160
  • Human:    1:2,200

A PUREPROVE®-based matrix removes more than 90% of the human background DNA. The final bacterial plus fungal/human DNA ratio is significantly higher in favor of the first, which substantially increases the sensitivity of downstream PCR methods and simultaneously reduces the time-to-result.

3. How does LOOXSTER® concentrate fungal DNA?

The determining variable for the binding protein is the number if unmethylated CpG motives (A/T[CpG]A/T). The number of these motives is significantly lower in fungal/bacterial than in human genomes. Therefore, fungal targets can clearly be separated from human genomes.

Application

4. What are the typical applications of LOOXSTER® universal?
Any applications that require the specific isolation and concentration of bacterial and fungal genomic DNA from various samples (food, environment, microbiological QC test such as swabs, etc.)
5. Do you recommend a special method for the detection of the bacterial DNA?

That depends on the application. If you want to detect pathogens causing severe infections especially sepsis we recommend the complete system VYOO® which incorporates the LOOXSTER® sample preparation.
In case you aim at detecting other bacteria that sepsis specific, LOOXSTER® can be used with any detection method as Southern blotting, restriction enzyme digestion, DNA microarrays, sequencing, genotyping, etc.

6. Which DNA volume is required for the LOOXSTER® protocol?
The volume used to resuspend the DNA at the end of the LOOXSTER® protocol depends upon the amount of DNA present. For example, a 5 ml blood sample extracted with a commercial DNA isolation kit and then applied to LOOXSTER® will result in ca. 3 µg DNA.
The resuspension volume depends upon the use of the DNA. In a classic PCR, the whole amount of DNA could be used in a reaction if needed. Here, the resuspension volume would depend upon the PCR final volume (50 µl, 25 µl).  In a real time PCR, an average of 100-250ng total DNA work well (No more than a maximum of 500 ng should be used!). Here too, the resuspension volume depends upon the final volume of the PCR reaction. However, we would not recommend using less than 10 µl buffer to resuspend the ca. 3 µg DNA.
7. What are the limitations of LOOXSTER® universal?

100% separation of bacterial/fungal from human DNA is not obtained. It is therefore recommended to account for and include controls for eukaryotic DNA in down-stream applications such as running the reaction with prokaryote specific and GAPDH primers in a (real-time) PCR.

8. Can LOOXSTER® universal be used for diagnostic purposes?
No, LOOXSTER® is designed and sold for R&D purposes only.

 

Performance

9. How long does it take to perform the whole LOOXSTER® universal protocol?

It takes 45 minutes to perform the entire protocol.

10. What are the advantages of LOOXSTER® universal over conventional bacterial genomic DNA isolation methods?

Other methods are not specific for bacterial and fungal genomic DNA when not performed with pure bacterial cultures. LOOXSTER® is a kit specially designed to concentrate bacterial and fungal genomic DNA from samples containing both prokaryotic and eukaryotic DNA. It is also possible to separate bacterial and fungal genomic DNA from inhibitors.

11. Can LOOXSTER® universal be used to concentrate bacterial and fungal genomic DNA from mixed species total genomic DNA?

Yes, LOOXSTER® universal was developed as a means to specifically separate bacterial and fungal genomic DNA from other sample components such as human background material, inhibitors, etc.

12. What kind of sample origins can be used for the LOOXSTER® universal protocol?

The LOOXSTER® universal does not depend upon the source of the materials or the isolation protocol/ kit used, as long as they result in total DNA preparations containing both prokaryotic and eukaryotic DNA

13. Does the total genomic DNA need pre-treatment before its application on the LOOXSTER® universal columns?

No. Genomic DNA obtained by various methods can be used with LOOXSTER®
universal without prior treatment as long as the DNA is resuspended in buffer which does not contain chaotropic, detergents or chelating agents.

14. Why is the human background DNA not removed completely?

Small amounts of human DNA in the concentrated sample proved to have a positive effect on subsequent pathogen detection.

15. How can the DNA content after LOOXSTER® universal elution be measured?
DNA concentration and purity can be determined by measuring the absorbance at 260 nm (A260) and 280 nm (A280) in a spectrophotometer using a quartz cuvette or a Nanodrop® spectrophotometer. Samples should be diluted in a low-salt buffer with neutral pH (e.g. Tris•HCl, pH 7.0). However, due to the minute prokaryotic DNA content of certain samples, it is possible to obtain results in down-stream applications (e.g. PCR) while DNA is not measurable using conventional methods.
16. Is it possible to interrupt the protocol?
It is not advisable to stop before the end of this short protocol.
17. Can LOOXSTER® columns be re-used?
SIRS-Lab recommends not to re-use the columns. DNAse free conditions and absence of traces of bacterial DNA cannot be guaranteed for columns previously used.


Validation 

18. Where can clinical studies and publications about LOOXSTER® universal be found?

For references and publications, please see our download section and publication list at the SIRS-Lab web sites. Additional publications are in progress.

19. How where the acceptance criteria established? How are false positive results excluded?

Threshold:
The cut off threshold of the amplicons was set at  <15 copies per single reaction. In the course of the technical validation, not more than 6.6 copies have been observed.
DNase free:
Further, all reagents and devices are DNase and Cell free.

These are only two factors in SIRS-Lab’s strict quality control program.

20. Have studies with spiking experiments been performed?

Spiking experiments have been performed. Nevertheless, spiked samples do not allow the same validity as clinical studies. Therefore SIRS-Lab relied on clinical samples to reach highest clinical validity.
 

Kit Properties

21. What does the LOOXSTER® universal kit contain?

LOOXSTER® universal is available in two sizes of 20 or 100 concentrations.

  • Concentration matrix
  • Equilibration and Washing Buffer
  • Elution Buffer
  • Column
  • Tubes
  • Manual
22. How should LOOXSTER® universal be stored and for how long?

LOOXSTER® universal's performances are guaranteed for 6 months when stored under proper conditions, that is matrix and buffers at 4°C, the other components at room temperature.

23. Is the concentration matrix for LOOXSTER® universal sold separately?

No, the LOOXSTER® universal concentration matrix is not sold separately.

24. Which additional material is needed to conduct experiments with LOOXSTER® universal?

No specific equipment is required to use LOOXSTER® universal besides common laboratory apparatus and reagents (ethanol, etc.). Please see our detailed reagents list in our manual.

25. Which costs, especially with regards to reagents, are to be expected for the complete experiment (from total genomic DNA isolation till pathogen detection)?

The costs are dependent upon the up- and down-stream applications that are planned in conjunction with LOOXSTER® universal.

Ordering and Support

26. Where can LOOXSTER® universal be ordered?

LOOXSTER® universal can be directly ordered at SIRS-Lab
by e-mail: products[at]sirs-lab.com
phone     +49 3641 579 729
fax:         +49 3641 508 432 

27. How can I contact SIRS-Lab for technical questions regarding LOOXSTER® universal?

e-mail:  support[at]sirs-lab.com
phone: +49 3641 579 729
fax:      +49 3641 508 432