Enterovirus + Herpes simplex (HSV1 + HSV2) + Varicella-Zoster virus (VZV) / Neisseria meningitidis + Streptococcus pneumoniae + Listeria monocytogenes PCR Kit CE/IVD

1 Intended use:
RealCycler MEBAVI is an in vitro diagnostic kit of reagents which allows real-time PCR qualitative detection of DNA from Neisseria meningitidis, Streptococcus pneumoniae, Listeria monocytogenes, Herpes Simplex Virus (type 1 + type 2), Varicella-Zoster Virus and RNA from Enterovirus in clinical samples.
The system includes an internal control CHIC (Competitive Heterologous Internal Control) to prevent false negatives due to reaction inhibition.

2 Principle of the test
The polymerase chain reaction (PCR) is based on the amplification of a specific region of the DNA/RNA by using complementary primers to the target sequence. Real-time PCR uses marked probes with fluorophores that emit fluorescence in the case of amplification. The cycle of the PCR protocol in which appears significant fluorescence is proportional to the DNA/RNA quantity present in the sample. This value is called Cycle Threshold (Ct) or Cycle Quantification (Cq).

In the MENELI amplification, Neisseria meningitidis is detected in the corresponding channel of FAM fluorophore, Streptococcus pneumoniaeis detected in the corresponding channel of TxR fluorophore, Listeria monocytogenesis detected in the corresponding channel of Alx647 or Cy5 fluorophore and the internal control is detected in the corresponding channel of Alx532 or HEX fluorophore.

In the ENHEVA amplification, Enterovirus is detected in the corresponding channel of FAM fluorophore, Herpesvirus (type 1 + type 2) is detected in the corresponding channel of TxR fluorophore, Varicella-Zoster virus is detected in the corresponding channel of Alx647 or Cy5 fluorophore and the internal control is detected in the corresponding channel of Alx532 or HEX fluorophore

3 Technical specifications
Sensitivity:
- Neisseria meningitidis: 10 copies/μL,
- Streptococcus pneumoniae: 100 copies/μL
- Listeria monocytogenes: 1 copy/μL.
- Enterovirus: 1 copy/μL.
- Herpesvirus type 1: 10 copies/μL.
- Herpesvirus type 2: 100 copies/μL.
- Varicella-Zoster virus: 100 copies/μL
The analytical sensitivity has been determined by limit dilution. This sensitivity has been showed in repeated assays with reproducibility over 95%.

Specificity:
- Neisseria meningitidis: ctrA gene.,
- Streptococcus pneumoniae: ply gene.
- Listeria monocytogenes: hlyA gene.
- Enterovirus: 5’ UTR region.
- Herpesvirus type 1: US6 gene.
- Herpesvirus type 2: DNA polymerase gene.
- Varicella-Zoster Virus (Single-stranted DNA-binding protein gene (ORF 29)).
Specificity validation has been performed according to experimental assays and BLAST analysis (www.ncbi.nlm.nih.gov/blast).

4. Contents
RealCycler MEBAVI includes the MENELI AmpliMix and ENHEVA AmpliMix, a retrotranscriptase, a MENELI DNA Positive control, which contains a mixture of Neisseria meningitidis, Streptococcus pneumoniae and Listeria monocytogenes and an ENHEVA Positive Control, which contains a mixture of Enterovirus RNA and Herpes Simplex virus and Varicella-Zoster virus DNA.
All reagents are ready to use without adding or rebuilding any component.