HHHH v.6
1 Intended use
RealCycler HHHH is an in vitro diagnostic kit of reagents which allows
real-time PCR qualitative detection of DNA from Herpesvirus type 1
(HSV1), Herpesvirus type 2 (HSV2), Varicella-Zoster Virus (VZV), EpsteinBarr Virus (EBV), Cytomegalovirus (CMV) and Herpesvirus type 6 (HHV6)
simultaneously in clinical samples, using SmartCycler® (Cepheid®). The
system includes an internal control of amplification to prevent false
negatives.
2 Technical specifications
- Sensitivity: 10 copies/µL Herpesvirus type 1, 10 copies/µL Herpesvirus
type 2, 10 copies/µL Varicella-Zoster Virus, 10 copies/µL Epstein-Barr
Virus, 1 copy/µL Cytomegalovirus and 10 copies/µL Herpesvirus type 6.
- Specificity: Herpesvirus type 1 (US4 y US5 genes), Herpesvirus type
2 (DNA polimerase gene), Varicella-Zoster Virus (Single-stranted
DNA-binding protein gene (ORF 29)), Epstein-Barr Virus (LMP1 gene),
Cytomegalovirus (Glycoprotein B gene) and Herpesvirus type 6 (U67
gene).
3 Stability and storage
All components of the RealCycler HHHH kit should be stored at -20ºC.
All reagents of the kit are stable at -20ºC until expiration date (see the
external label).
4 Principle of the test
The polymerase chain reaction (PCR) is based on the amplification of a
specific region of the pathogen genome by using specific primers and
fluorescent dyes. Real-time PCR uses marked probes with fluorochromes.
There is an emission of fluorescence that is proportional to the quantity of
present DNA in the clinical sample. The Cycle threshold (Ct) is the cycle
of the PCR protocol in which appears significant fluorescence. In the
amplification of HSV1/HSV2, Herpesvirus type 1 is detected in FAM,
Herpesvirus type 2 in TxR and the internal control in Alx532. In the
amplification of VZV, Varicella-Zoster Virus is detected in FAM and the
internal control in Alx532. In the amplification of EBV/CMV, Epstein-Barr
Virus is detected in FAM, Cytomegalovirus in TxR and the internal control
in Alx532. In the amplification of HHV6, Herpesvirus type 6 is detected in
FAM and the internal control in Alx532.
5 Contents
RealCycler HHHH includes the HV12 MasterMix, VZV MasterMix, CMEB
MasterMix and HHVI MasterMix dispensed into amplification tubes, a
HSV1 DNA Positive Control, a HSV2 DNA Positive Control, a VZV DNA
Positive Control, an EBV DNA Positive Control, a CMV DNA Positive
Control and a HHV6 DNA Positive Control. All reagents are ready to use
without adding or rebuilding any component.
Component
Vials
Num.
Volume
Storage
HV12
MasterMix
6
21 µL
-15 /-25 ºC
VZV
MasterMix
6
18,2 µL
-15 /-25 ºC
CMEB
MasterMix
6
19,5 µL
-15 /-25 ºC
HHVI
MasterMix
6
19 µL
-15 /-25 ºC
HSV1 DNA
Positive
Control
1
30 µL
-15 /-25ºC
HSV2 DNA
Positive
Control
1
30 µL
-15 /-25 ºC
VZV DNA
Positive
Control
1
30 µL
-15 /-25ºC
EBV DNA
Positive
Control
1
30 µL
-15 /-25ºC
CMV DNA
Positive
Control
1
30 µL
-15 /-25ºC
HHV6 DNA
Positive
Control
1
30 µL
-15 /-25ºC
6 Additional materials and equipment required
and not supplied
- Real-time PCR system SmartCycler® (Cepheid®)
- Microcentrifuge for SmartCycler® tubes
- SmartCycler® tube racks
- DNA isolation kit
- Disposable gloves
- Adjustable pipettes
- Pipette tips with filter
- Freezer(-20ºC)
7 Warnings and Precautions
- All components of the kit must be kept cold while you are working.
- Load tubes in the SmartCycler® immediately after adding DNA.
- Do not expose tubes with MasterMix to light for a long time.
- To defrost and to freeze repeatedly the reagents can decrease the
sensitivity of the kit.
- Use disposable gloves.
- Use adequate and calibrated pipettes and pipette tips with filter.
- The tests must be carried out by qualified personnel and following good
laboratory practices.
- Do not use the kit after the expiry date.
- For in vitro diagnostic use. CE mark doesn't currently include the
detection of Cytomegalovirus. The results obtained must be considered
only for clinical research purposes.
- Cepheid® and SmartCycler® are trademarks of Cepheid Corporation.
8 Clinical samples
- Collect samples in sterile tubes.
- Storage and transportation frozen at -20ºC until use.
- Use well purified DNA free of PCR inhibitors.
d) Results
FAM
Std/Res
TxR
Std/Res
Alx532
Std/Res
Alx532
Ct
Interpretation
POS
ND
Indifferent
Indifferent
HSV1 POSITIVE
ND
POS
Indifferent
Indifferent
HSV2 POSITIVE
POS
POS
Indifferent
Indifferent
HSV1 and HSV2
POSITIVE
ND
ND
POS
ND
ND
POS
Ct out of
range
NOT ASSESSABLE
ND
ND
ND
0,00
NOT ASSESSABLE
Ct within the
NOT DETECTED
range
Varicella-Zoster virus (blue mark)
b) Protocol
9 Procedure
a) DNA extraction
Herpesvirus type 1 and 2 (white mark)
b) Protocol
c) PCR reaction set-up
- Thaw MasterMix tubes necessary to amplify the clinical samples and
VZV DNA Positive Control.
- Add 9,5 µL of DNA sample or control to each reaction tube.
- Spin tubes to transfer MasterMix to optical area of tube. Check there are
no bubbles in the optical area.
- Load tubes in the SmartCycler®.
- Create Run > Dye Set > FATA25
- Add/Remove Sites: assign both the“VZV” protocol and sites to each
sample including DNA Positive Control.
- Start Run.
c) PCR reaction set-up
- Thaw MasterMix tubes necessary to amplify the clinical samples and
HSV1 and HSV2 DNA Positive Controls.
- Add 7 µL of DNA sample or control to each reaction tube.
- Spin tubes to transfer MasterMix to optical area of tube. Check there are
no bubbles in the optical area.
- Load tubes in the SmartCycler®.
- Create Run > Dye Set > FATA25
- Add/Remove Sites: assign both the“HV12” protocol and sites to each
sample including DNA Positive Control.
- Start Run.
d) Results
FAM
Std/Res
Alx532
Std/Res
Alx532
Ct
Interpretation
POS
Indifferent
Indifferent
VZV POSITIVE
ND
POS
ND
POS
Ct out of
range
NOT ASSESSABLE
ND
ND
0,00
NOT ASSESSABLE
Ct within the
NOT DETECTED
range
Cytomegalovirus and Epstein-Barr Virus
(red mark)
Herpesvirus type 6
b) Protocol
b) Protocol
c) PCR reaction set-up
c) PCR reaction set-up
- Thaw MasterMix tubes necessary to amplify the clinical samples and
EBV and CMV DNA Positive Controls.
- Add 8 µL of DNA sample or control to each reaction tube.
- Spin tubes to transfer MasterMix to optical area of tube. Check there are
no bubbles in the optical area.
- Load tubes in the SmartCycler®.
- Create Run > Dye Set > FATA25
- Add/Remove Sites: assign both the“CMEB” protocol and sites to each
sample including DNA Positive Control.
- Start Run.
- Thaw MasterMix tubes necessary to amplify the clinical samples and
HHV6 DNA Positive Control.
- Add 8,5 µL of DNA sample or control to each reaction tube.
- Spin tubes to transfer MasterMix to optical area of tube. Check there are
no bubbles in the optical area.
- Load tubes in the SmartCycler®.
- Create Run > Dye Set > FATA25
- Add/Remove Sites: assign both the “HHVI” protocol and sites to each
sample including DNA Positive Control.
- Start Run.
d) Results
d) Results
FAM
TxR
Alx532
Std/Res Std/Res Std/Res
Alx532
Ct
Interpretation
FAM
Std/Res
Alx532
Std/Res
Alx532
Ct
Interpretation
Indifferent
HHV6 POSITIVE
POS
ND
Indifferent
Indifferent
EBV POSITIVE
POS
Indifferent
ND
POS
Indifferent
Indifferent
CMV POSITIVE
ND
POS
POS
POS
Indifferent
Indifferent
EBV and CMV
POSITIVE
ND
POS
ND
ND
POS
Ct within
NOT DETECTED
the range
ND
ND
ND
ND
POS
Ct out of
range
NOT
ASSESSABLE
ND
ND
ND
0,00
NOT
ASSESSABLE
Ct within
the range
Ct out of
range
0,00
NOT DETECTED
NOT ASSESSABLE
NOT ASSESSABLE
e) Example result
Herpesvirus type 1 and 2
Figure 1 (Channel FAM: HSV1): Result obtained to amplify a negative
control, a HSV1 positive control and a HSV2 positive control. Negative
control (A11 blue): not detected. HSV1 positive control (A15 orange): Ct
= 32,88. HSV2 positive control (A12 grey): not detected.
Figure 2 (Channel TxR: HSV2): Result obtained to amplify a negative
control, a HSV1 positive control and a HSV2 positive control. Negative
control (A11 blue): not detected. HSV1 positive control (A15 orange): not
detected. HSV2 positive control (A12 grey): Ct=30,72.
Varicella-Zoster virus
Figure 1 (Channel FAM:VZV): Result obtained to amplify a negative control
and VZV positive control. Negative control (A11 blue): not detected. VZV
positive control (A12 grey): Ct = 30,80.
Figure 2 (Channel Alx532: internal control): Result obtained to amplify a
negative control and VZV positive control. Negative control (A11 blue):
Ct = 32,67. VZV positive control (A12 grey): Ct = 31,99.
Cytomegalovirus and Epstein-Barr Virus
Figure 3 (Channel Alx532: internal control): Result obtained to amplify a
negative control, a HSV1 positive control and a HSV2 positive control.
Negative control (A11 blue): Ct= 33,09. HSV1 positive control (A15
orange): Ct=32,80. HSV2 positive control (A12 grey): Ct=33,46.
Figure 1 (Channel FAM: EBV): Result obtained to amplify two negative
controls, an EBV positive control and a CMV positive control. Negative
control (A3 blue): not detected, negative control (A6 grey): not detected.
EBV positive control (A7 orange): Ct = 29,37. CMV positive control (A8
green): not detected.
Figure 2 (Channel TxR: CMV): Result obtained to amplify two negative
controls, an EBV positive control and a CMV positive control. Negative
control (A3 blue): not detected, negative control (A6 grey): not detected.
EBV positive control (A7 orange): not detected. CMV positive control (A8
green): Ct = 28,57.
Figure 2 (Channel Alx532: internal control): Results obtained in the
amplication of two negative controls and one HHV6 positive control.
Negative control (A9 blue): Ct = 28,58. Negative control (A10 grey): Ct =
29,46. HHV6 positive control (A11 orange): Ct = 27,66.
10 Quality control
It is recommended to carry out both positive control and negative control
whenever an analysis is realized.
To validate the results, Ct's values obtained in the positive control and the
internal controls must be between the ranges specified in the internal label
of the kit.
Every lot of RealCycler HHHH kit has been tested according to the
specifications of the real-time PCR using the SmartCycler® device
(Cepheid®).
Date of publication: October, 2011.
Figure 3 (Channel Alx532: internal control): Result obtained to amplify two
negative controls, an EBV positive control and a CMV positive control.
Negative control (A3 blue): Ct = 29,74, negative control (A6 grey): Ct =
29,71. EBV positive control (A7 orange): Ct = 29,14. CMV positive control
(A8 green): Ct = 29,08.
Herpesvirus type 6
Figure 1 (Channel FAM: HHV6): Results obtained in the amplication of two
negative controls and one HHV6 positive control. Negative control (A9
blue): not detected. Negative control (A10 grey): not detected. HHV6
positive control (A11 orange): Ct = 30,09.