FAQ VYOO^® Sepsis Test

1. What is VYOO^®?

VYOO^® is a multiplex PCR assay for the detection of 34 bacterial and 6 fungal species as well as 5 antibiotic resistance genes in complex samples. The whole process takes 6 hours.

Principle

2. What is the principle of VYOO^®?

The multiplex PCR protocol is designed for the highly specific amplification of a series of bacterial and fungal targets. The prior concentration of target-DNA is realized with SIRS-Lab’s proprietary PUREPROVE^® technology incorporated in LOOXSTER^®. This sample preparation system is an integral part of VYOOTM. Read more about LOOXSTER^® here. After the sample preparation, the bacterial and fungal targets are detected by PCR. The following analysis of the generated amplicons is processed by gelelectrophoretic techniques.

Application

3. What are the typical applications of VYOO^® and what kind of sample origins can be applied?

The primer selection of VYOO^® is designed for the detection of sepsis pathogens and the test has been validated for whole blood samples. However, the test was applied to alternative body fluids, e. g. ascites fluids of patients of spontaneous bacterial peritonitis (see study), and is attributed to all kinds of samples, in which bacterial and fungal pathogens are expected with a high concentration of human background DNA.
The test can be used for samples containing causative pathogens of other systemic infections. If you have possible application fields for the test, please contact us for specifically designed test kits.

4. How many species and resistance genes can be detected?

VYOO^® detects 36 bacterial and 6 fungal species known to cause sepsis. In the same process, 5 important antibiotic resistance genes are identified. Overall, 99% of sepsis-relevant species are covered. View the complete species list below.

5. Which kind auf pathogen cells/PCR targets are captured by VYOO^®?

The assay detects nucleic acids of:

• whole and lyzed cells
• intracellular, adherent, and phagocytized cells
• non-viable and non-culturable cells
• cells which are affected by antibiotic pre-treatment.

A differentiation of dead and viable cells is not possible.

6. Can VYOO^® be used for diagnostic purposes?

No, VYOO^® is designed and sold for R&D purposes only.

7. What are the advantages of VYOO^® over alternative NAT detection tools?

Only standard microbiology laboratory equipment is necessary for performing the VYOO^® protocol (e.g. centrifuges, incubators). There is no special training required and usual PCR skills are sufficient for the performance of the whole protocol including data analysis.

Standard equipment for VYOO^®:

• mechanical cell lysis step with for instance a VORTEX Genie II shaker with low budget tube fixtures
• centrifugation steps with conventional centrifuges for 2 ml – 30 ml tubes
• multiplex PCR with standard PCR thermocyclers
• result analysis by standard gel electrophoresis equipment with common gel sizes

8. What are the advantages of VYOO^® over current blood culture gold standard?

The clinical standard for the detection of sepsis pathogens is the blood culture technique. VYOO^® is designed as extension to current methods.
80-90% of all blood cultures of patients suspected of systemic infections are negative. Further drawbacks of culture based methods are low abundance of causative agents, non-cultivable organisms are not detected, and time to result of 3 to 5 days.

Compared to blood culture, VYOO^® has the following advantages:

• time-to-result: 6 hours
• detection of non-cultivable organisms
• unaffected by antibiotic pre-treatment

Performance

9. How long does it take to perform the whole VYOO^® protocol from sample to result?

The process starting from samples as e.g. whole blood takes 6 hours.

10. How many samples can be tested simultaneously?

The number of samples which can be tested simultaneously is restricted to the facilities for shaking and centrifugation. In the DNA enrichment step (with LOXXSTER^®) 16 samples can be handled at a time. The number of multiplex PCR samples (3 tubes per sample, inclusive one inhibition control per VYOO^® primer pool) is limited to the sample slots of the used thermocycler.

11. Which education is necessary for performance of VYOO^® from sample to result?

There is no special training required.

12. What are the limitations of VYOO^®?

The primer assay is designed for the detection of sepsis pathogens. In case of bacteria, the assay is limited to 34 sepsis specific targets (November 2007). Fungi are detected in the same assay but in an open 18S approach. That means that there are not limitations in the identification of species.

Generally, the VYOO^® technology can be applied in various cases of severe infections. For special requests and designed assays please contact our product management: products[at]sirs-lab.com

13. Is the threshold level for all targets identical and how is the level?

The strength of the target specific band in gelelectrophoretic amplicon analysis corresponds to the length of the target sequence, the genome number (pathogen count within the sample), and to the target copy number within the pathogen genome. The threshold number of the VYOO^® targets therefore varies between 1 and 4,200 genome copies per sample.

14. Is VYOO^® liable for contamination?

The test’s liability for contamination is relatively low. No special clean room is required. Be aware of the fact that the main cause for contaminations with foreign DNA is insufficiencies while sampling.
Ensure that the intake of foreign DNA targets is excluded. It is recommended to use non-powdered nitrile gloves, filter tips and equipment that has not been in contact with any possible bacterial DNA contamination sources. You should avoid contamination with DNases and prepare/pipette the PCR mixes in a bacterial DNA free area. Use water and solutions that are free from bacterial and fungal DNA.

15. Does the total genomic DNA need pre-treatment before the application of VYOO^®?

No. Genomic DNA obtained by various methods can be used with VYOO^® multiplex PCR without prior treatment as long as the DNA is resuspended in buffer which does not contain chaotropic, detergent or chelating agents. VYOO^® contains all buffers and consumables for the isolation of total DNA from complex samples.

16. How can the DNA content after LOOXSTER^®-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.

17. Is it possible to interrupt the protocol?

It is strictly recommended to minimize the storing time for isolated total DNA or enriched pathogen DNA fractions. If it is impossible to complete the protocol dissolve your purified DNA after precipitation over night at 4°C (after whole DNA isolation or the pathogen DNA concentration step) or disrupt the protocol after the respective DNA aqueous dissolutions by storing the samples at –80°C.

Validation 

18. Is VYOO^® a CE certified diagnostic product?

The CE certification is currently under progress.

19. Does VYOO work with an optimized primer Tm?

We achieve highest PCR sensitivity by an optimized primer Tm with minimal deviations.

20. How was the sensitivity of the assays measured?

SIRS-Lab checked every primer pair with regard to other pathogen amplicons, which are not part of the assay. Further the primers have been tested against each other. In a whole more than 150,000 single reactions have been performed to create a highly specific 47 plex assay.

21. What is the sensitivity expressed in CFU/ml?

The sensitivity of VYOO lies in a range of 1-3 CFU/ml. However, this is a question of how analytical sensitivity is measured. A direct correlation between CFU/ml and PCR based methods is only of restricted meaning. The CFU only covers cultivable germs. PCR methods are not limited to the detection of cultivable organisms. That means that the gold standard blood culture is limited compared to PCR based methods which reach higher sensitivity than the CFU expresses.

22. Where to find clinical studies and publications about VYOO^®?

For references and publications, please consider the publications section on the SIRS-Lab web sites. Application and technical notes can be downloaded here:

Kit Properties

23. What does a VYOO^® kit contain?

• buffers and tubes with glass beads for the mechanical cell lysis step
• all components for DNA isolation (tubes with matrix, buffers)
• tubes, cartridges with concentration matrices, and solutions for the enrichment of pathogen DNA
• multiplex PCR primers (divided into two pools in 0.2 ml tubes)
• inhibition control DNA, amplicon specific DNA ladders, gel loading buffer
• elaborate manual

24. Which additional material is needed to conduct experiments with VYOO^®?

• Multiplex PCR mix (incl. multiplex PCR-buffer, dNTPs and DNA polymerase, e. g. QIAGEN Multiplex PCR Kit, Cat.-No. 206143 or 206145)
• Isopropanol
• 3 M NaAc pH 5,2, Ethanol (70%)
• molecular biology grade water (sterile, DNA- and DNase-free), e. g. from MINERVA (Water, PCR-Grade, UV-treated).

Please refer to the manual for further information.

25. How should VYOO^® be stored and for how long?

VYOO^® performances are guaranteed for 6 months when stored under proper conditions: LOOXSTER^®-matrix and buffers at 4°C, the other components at room temperature.

Ordering and Support

26. Where can VYOO^® be ordered?

VYOO^® can directly be ordered from SIRS-Lab:
Key Account Management
Email:      products[at]sirs-lab.com
Phone:     +49 3641 579 729
Fax:        +49 3641 508 432

27. How can SIRS-Lab be contacted for technical questions regarding VYOO^®?

SIRS-Lab’s technical service can be contacted:
Email:     support[at]sirs-lab.com
Phone:    +49 3641 579 729
Fax:       +49 3641 508 432