HPV Direct Flow CHIP Kit

HPV Direct Flow
CHIP Kit
Screening and genotyping of human
papillomavirus based on PCR amplification
and reverse dot blot hybridization
30 determinations
Ref. MAD-003930M
Produced under UNE-EN 375 regulations
For in vitro diagnostic use
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
1
Rev: 2014-09
TABLE OF CONTENTS
1. INTENTED USE ........................................................................................................................... 3
2. TEST PRINCIPLE ......................................................................................................................... 3
3. REAGENTS INCLUDED IN THE KIT ............................................................................................. 4
4. STORAGE AND STABILITY ......................................................................................................... 4
5. EQUIPMENT AND MATERIALS REQUIRED BUT NOT SUPPLIED IN THE KIT ............................. 5
6. REMARKS AND PRECAUTIONS ................................................................................................. 5
7. OPERATING PROCEDURE .......................................................................................................... 6
7.1. REAGENTS PREPARATION ..................................................................................................... 6
7.2. SAMPLE PREPARATION FOR DIRECT PCR.............................................................................. 6
7.3. PCR REACTION ....................................................................................................................... 8
7.4. AUTOMATIC FLOW-THROUGH REVERSE HYBRIDIZATION ................................................... 8
8. INTERPRETATION OF THE RESULTS ........................................................................................ 10
9. REPRODUCIBILITY OF HPV Direct Flow CHIP ......................................................................... 11
10. ANALITICAL SENSIBILITY AND SPECIFICITY OF HPV Direct Flow CHIP................................. 11
11. TROUBLESHOOTING ............................................................................................................. 11
12. BIBLIOGRAPHY ...................................................................................................................... 12
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
2
Rev: 2014-09
1. INTENTED USE
HPV Direct Flow CHIP is a diagnostic kit for in vitro detection of human papillomaviruses (HPV).
HPV detection has become a very important tool in diagnostic because infection with these
viruses constitutes an essential factor for cervical and anogenital carcinogenesis (zur Hausen et
al, 1974; Walboomer et al, 1999; zur Hausen, 1996; zur Hausen 2002).
2. TEST PRINCIPLE
Based on their association with different lesion grades, HPV have been classified as (Muñoz
2003) high risk HPVs or oncogenic, that can induce carcinogenesis; and low risk HPVs, that
cause genital warts and collaborate with high risk HPVs.
HPV Direct Flow CHIP is intended for simultaneous screening and genotyping of 36 HPV types
(High risk- HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82-, and low
risk- HPV 6, 11, 40, 42, 43, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, 81, 84 and 89 (=CP6108)) by
PCR (polymerase chain reaction), followed by reverse dot blot automatic hybridization, based
on DNA-Flow Technology (e-BRID System®). Clinical samples (fresh and paraffin embedded) are
amplified directly, without the need of DNA extraction.
This kit is based on the amplification of a human papillomavirus L1 consensus region by PCR
and hybridization to specific DNA probes immobilized onto a nylon membrane. The DNA-Flow
based automatic hybridization platform allows the binding of the amplified DNA to the
complementary capture probes in a three-dimensional porous environment, which enables a
very fast coupling between the PCR product and its specific probe. Biotinilated PCR products
are hybridized with specific probes and the hybridization signal is developed by a colorimetric
immunoenzimatic reaction (Streptavidin-Alkaline Phosphatase and NBT-BCIP chromogen). The
substrate-chromogen reaction generates a dark-purple precipitate in the position where the
specific probe has hybridized with the PCR amplicon and this signal is automatically captured
and analyzed (e-BRID System®). This technology has a very high sensitivity for HPV detection
and it can be performed in a very short time comparing to other systems, reducing total
processing time from hours to minutes.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
3
Rev: 2014-09
3. REAGENTS INCLUDED IN THE KIT
This kit includes all necessary reagents for direct PCR amplification and hybridization of 30
clinical samples.
- Reagents for direct PCR:
PCR Mix (1x 1700 µl)
Phire® Hot Start II DNA polymerase* (1 x 30 µl).
- Reagents for Reverse dot blot hybridization:
Reagent A:
Reagent B:
Reagent C:
Reagent D:
Reagent E-1:
Reagent E-2:
Reagent F:
HPV CHIP
a
Hybridization solution
Blocking solution
Streptavidin-Alkaline Phosphatase
Washing buffer I
Substratea
Chromogena
Washing buffer II
Spotted membranes
90 ml
35 ml
18 ml
65 ml
10 ml
10 ml
50 ml
30 units
The developing solution must be prepared just before use by mixing 1:1 the reagents E-1 and E-2.
* Trademark and license statements
Notice to Purchaser: Limited license the purchase price of this product includes a limited, nontransferable license under U.S. and foreign patents (5,500,363 and 5,352,778) owned by New England
Biolabs, Inc. to use this product. No other license under these patents is conveyed expressly or by
implication to the purchaser by the purchase of this product.
This product is licensed under US Patent 5,436,149 owned by TaKaRa Shuzo Co. Ltd. This product is sold
under license from Affibody AB, Sweden. Phire® and DyNAzyme™ are trademarks or registered
trademarks of Finnzymes Oy, a Thermo Fisher Scientific company. Affibody® is a registered trademark of
Affibody AB, Sweden.
4. STORAGE AND STABILITY
PCR reagents: Shipped at 2-8 ºC and then stored at -20ºC after reception. Thaw on ice just
before use. Reagents are stable until expiration date. These reagents must be stored isolated
from any source of contaminating DNA (e.g. PCR products).
Hybridization reagents: Shipped and stored at 2-8 ºC. Do not freeze. Reagents and HPV CHIPs
are stable until expiration date. Developing solution must be prepared just before use.
Hybridization reagent should be brought to 41ºC before use and all other hybridization
reagents should be used at room temperature (20-25ºC).
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
4
Rev: 2014-09
5. EQUIPMENT AND MATERIALS REQUIRED BUT NOT SUPPLIED IN THE KIT
Equipment:
 Thermocycler.
 Microcentrifuge.
 Thermostatic bath or heating block.
 e-BRID System®.
Consumables:
 Disposable gloves.
 Disposable sterile pipette tips.
 Sterile PBS buffer (DNase/RNase-free).
 PCR DNase/RNase-free Eppendorf tubes 0.2/ 0.5/1.5 ml.
 Distilled water.
 PARAFFIN TISSUE PROCESSING KIT, Ref: MAD-003952M (30 tests).
6. REMARKS AND PRECAUTIONS
During the amplification and hybridization of the samples it is recommended to use disposable
gloves. The most common source of contamination is the PCR product created in the
laboratory. In order to avoid this, it is very important to keep two different working areas: preand post-PCR. In the pre-PCR area, clinical samples are manipulated and added after adding
the Taq polymerase to the PCR tubes. Amplified products will be manipulated and hybridized
in the post-PCR area. These two zones must be physically separated and it is very important
not to share any material between both working stations (laboratory coats, pipettes, etc.). The
working flow must be in one way direction, from pre-PCR to post-PCR and never in the other
direction. This is important to avoid false positive cases due to contamination with PCR
products.
It is recommended to include negative controls (containing all the PCR components except the
DNA) during amplification.
7. OPERATING PROCEDURE
HPV Direct Flow CHIP kit has been optimized for direct use from clinical samples, without the
need of previous DNA extraction. Although not required, the kit can be used with purified
DNA.
The membranes are intended to be used only once. Do not touch the membranes with bare
hands and keep them away from any source of contamination.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
5
Rev: 2014-09
7.1. REAGENTS PREPARATION
All the reagents are supplied “ready to use”.
PCR MIX:
Thaw PCR Mix and Phire® Hot Start II DNA Polymerase before the first use and proceed as
follows:
-
Spin down the vial of Phire® Hot Start II DNA Polymerase for a few seconds.
Add 30 µl of Phire® Hot Start II DNA Polymerase to the PCR Mix.
Mix well by inverting the vial several times and centrifuge for a few seconds.
Dispense 54 µl aliquots of the mix into 30 0.2 ml PCR tubes.
The PCR tubes can be stored at 2-8ºC for one week or at -20ºC for 3 months.
7.2. SAMPLE PREPARATION FOR DIRECT PCR
-
Cytological swabs.
 Prepare one 1.5-2 ml eppendorf tube containing 400 µl of PBS buffer (repeat for
every sample). Dip the tip of the swab or brush into the PBS solution and squeeze
gently against the tube´s wall to detach the cells
 Centrifuge at 2000 rpm for 1 min. Remove the supernatant carefully.
 Resuspend the cell pellet in 25-50 l of PBS buffer (depending on the size of the
pellet).
 Use 6 l of this cell suspension as DNA template for the PCR reaction. The
remaining volume can be stored at 4ºC for one week or at –20ºC for 2 months.
-
Liquid-based cytology.
 Let the cells settle at the bottom of the vial. Place 150-200 µl of this cell
suspension in a 1.5- 2 ml tube.
 Centrifuge at 2000 rpm for 1 min. Remove the supernatant carefully.
 Wash the cell pellet by resuspending the cells in 400 l of PBS buffer. Centrifuge at
2000 rpm for 1 min. Remove the supernatant carefully.
 Resuspend the cell pellet in 25-50 l PBS buffer.
 Use 6 l of this cell suspension as DNA template for the PCR reaction. The
remaining volume can be stored at 4ºC for one week or at –20ºC for 2 months.
-
Paraffin embedded sections.
Although the system works properly with Direct PCR from paraffin tissue samples, sometimes,
it is difficult to guarantee the correct handling of the paraffin block and therefore it is
recommended to start from purified DNA to avoid any contamination.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
6
Rev: 2014-09

For DNA extraction from paraffin-embedded sections: please follow standard
purification methods. Use 6 µl of purified DNA as template for PCR.

For direct PCR from paraffin samples: it is recommended to use the reagents
included in the PARAFFIN TISSUE PROCESSING KIT (Ref: MAD-003952M) and follow
the protocol:
 Take 1-3 sections (depending on the tissue size) of 10 m thickness and
place them in a 0.5 ml Eppendorf tube using a needle or dissection
forceps. Note: remove as much rests of paraffin as possible from the tissue
sections.
 Add 400 µl of mineral oil.
 Heat the sample at 95ºC for 2 min.
 Centrifuge at 2000 rpm for 1 min and remove the remaining mineral oil.
 Add 60 l of Extraction Buffer and 1.5 µl of DNA Release to the tube.
 Heat the sample at 60ºC for 30 min and 98 ºC for 10 min (this procedure
can be performed in a thermocycler or a heat-block).
 Centrifuge at 2000 rpm for 1 min.
 The top of the liquid will contain the rests of paraffin and mineral oil from
the samples. Cell-debris will be pelleted at the bottom of the tube and the
DNA will be in the suspension under the paraffin layer. Use 6 l of this
liquid suspension as DNA template. The remaining sample can be stored at
4ºC for one week or at –20ºC for 2 months.
Notes:
 For tissue sections bigger than 1 cm2, it is recommended to increase the
volume of Extraction Buffer and DNA Release proportionally to assure the
complete dipping of the tissue.
 If after incubation the tissue has not been digested completely, it is
recommended to add additional volume of Extraction Buffer and DNA
Release and repeat incubation for 30 min.
 Direct PCR has not been assayed with other kind of clinical samples (i.e.
stained samples or cytological extensions). For these cases, DNA
purification is recommended.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
7
Rev: 2014-09
7.3. PCR REACTION
Add 6 µl of DNA template to a PCR mix tube (prepared as indicated in step 7.1). Place all the
tubes in the thermocycler and amplify the DNA under these conditions (PCR program):
1 cycle 98ºC
98ºC
5 cycles 42ºC
72ºC
98ºC
45 cycles 60ºC
72ºC
1 cycle 72ºC
8ºC
5 min*
5s
5s
10 s
5s
5s
10 s
1 min
∞
* When purified DNA is used, this initial denaturation step can be reduced from 5 min to 30 s.
Keep PCR products at 8-10ºC after the reaction is completed. Samples can be hybridized
immediately or stored in a post-PCR fridge at 8-10ºC for 1-2 days.
7.4. AUTOMATIC FLOW-THROUGH REVERSE HYBRIDIZATION
The hybridization procedure, images capture and analysis of results are performed
automatically by the e-BRID System®, supported by hybriSoftTM.
Set up the instrument following the instructions in the user manual (provided with the
instrument).
Before starting the automatic procedure:
1.
2.
3.
4.
5.
6.
7.
8.
Denature the PCR products by heating them at 95 ºC for 5 min in a thermocycler and
then cooling them on ice for at least 2 min.
Open the tubes caps and place the PCR tubes in the rack container of the e-BRID
System®.
Select the hybridization program and the number of tests to be assayed.
Input the samples identification
Verify the required volumes for all the reagents and place them at room temperature
in their corresponding sites.
Pre-warm Reagent A (Hybridization solution) at 41 ºC.
Place the HPV CHIPs in the indicated position of the chamber.
Prepare the required volume of developing solution by mixing reagents E1-E2 (1:1).
Place it in the corresponding position in the reagents´ rack.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
8
Rev: 2014-09
Automatic procedure:
a) Set the chamber temperature at 41ºC. Dispense 500 µl of pre-heated Reagent A
(Hybridization solution) into each HPV CHIP, incubate at 41 ºC for 3 min.
b) Remove the reagent by vacuum.
c) Mix 500 µl of preheated Reagent A (Hybridization solution) and 40 µl of each PCR
sample. Dispense both together into the HPV CHIP.
d) Incubate at 41 ºC for 5 min.
e) Remove the reagent by vacuum.
f) Perform 3 washes with 500 µl of pre-heated Reagent A (Hybridization solution).
g) Set the chamber temperature at 30 ºC.
h) Perform 2 incubations with 500 µl of Reagent B (Blocking solution) into each HPV
CHIP and incubate each time for 2 min 30 s at 30 ºC.
i) Remove the reagent by vacuum.
j) Dispense 500 µl of Reagent C (Streptavidin-Alkaline Phosphatase) into each HPV
CHIP. Incubate for 3 min at 30 ºC.
k) Remove the reagent by vacuum.
l) Set the chamber temperature at 36 ºC.
m) Perform 4 Washes with 500 µl of Reagent D (Washing buffer I).
n) When the chamber reaches 36 ºC, dispense 500 µl of Reagent E (developing
solution) into each one of the HPV CHIPs. Incubate at 36 ºC for 4 min.
o) Remove the reagent by vacuum.
p) Perform 3 washes with 500 µl of Reagent F (Washing buffer II) into each CHIP.
q) Image capture and analysis.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
9
Rev: 2014-09
8. INTERPRETATION OF THE RESULTS
The following figure shows the distribution of spots in the membrane:
B
33
58
42
71
16
52
B
B
35
59
43
72
18
53
6
69
C
39
66
44/
55
89
26
56
11
70
U
45
68
54
84
31
58
40
71
16
51
73
61
B
33
59
44/
55
72
18
52
82
62/
81
C
35
66
54
89
26
53
6
67
U
39
68
61
84
31
56
11
69
42
45
73
62/
81
B
40
70
43
51
82
67
The HPV CHIP contains a quality control that ensures the correct interpretation of the results.
All the membranes must be positive for the hybridization control (spots “B” in the figure
above). This signal indicates that the hybridization has worked correctly.
A housekeeping human gene fragment is co-amplified during the PCR as internal amplification
control (Spot “C”). A positive signal in it shows that the amplification worked efficiently and
that there was enough DNA template in the clinical sample. No signal detected in spot “C”
might indicate failures during amplification, low yield of DNA template or insufficient clinical
material.
Clinical samples positive for HPV infection will have a signal in the spot “B” (hybridization
control), spot “C” (PCR control), spot “U” (HPV Universal probe) and in the corresponding HPV
genotype probe, whereas clinical samples that are negative for HPV infection will have signals
only in spots “B” and “C”.
Some samples can show positivity for the hybridization control (“B”), PCR control (“C”) and
HPV Universal control (“U”) but not for any specific HPV probe, indicating that the sample is
positive for a HPV genotype not included in the CHIP detection panel.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
10
Rev: 2014-09
9. REPRODUCIBILITY OF HPV Direct Flow CHIP
Negativity of the samples. Clinical samples that were negative for HPV infection when tested
by HPV Direct Flow CHIP were re-analyzed by the same method demonstrating 100%
reproducibility in all the cases. Moreover, when re-analysis was performed starting from
purified DNA samples, negative results were confirmed.
Positivity of the samples. Reproducibility for detection of single and multiple HPV infection was
determined, obtaining 100% agreement in all the assays. Single infection reproducibility was
independently assayed by analysis of synthetic samples containing either HPV 16 or 18 (NIBSC
standards). For multiple infections, a panel of HPV 16, 18, 31, 52, 45, 59, 73, 6, 40 and 42 was
tested in 135 independent experiments, obtaining a 100% agreement between results.
10. ANALYTICAL SENSITIVITY AND SPECIFICITY OF THE HPV Direct Flow CHIP
Analytical sensitivity: Serial dilutions of HPV 16 and 18 WHO standard DNAs (NIBSC Institute)
were tested under a background of 20 ng genomic DNA. The detection limit was 1-10 copies of
viral genome equivalents.
Analytical specificity: No cross-reactivity was found between HPV genotypes included in the
test. In addition, reactivity with genital pathogens Herpes, Neiserria gonorrhoeae and
Chlamydia trachomatis was also excluded.
11. TROUBLESHOOTING
Observation
Approach
No signal in the hybridization control
Make sure all the reagents have been properly added
and repeat the assay.
No signal in the amplification control
Insufficient amount of DNA/clinical specimen or
inhibitors present in the sample. Repeat PCR
increasing input sample and/or use purified DNA
template.
HPV detection in the negative control
Contamination problems in pre-PCR area or in the
hybridization reagents. Clean the working areas,
repeat the PCR and the hybridization.
Weak hybridization signals
Check the expiry date of the solutions and storage
conditions. Repeat the PCR increasing the input DNA.
Confirm that the denaturation step is correctly
performed.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
11
Rev: 2014-09
12. BIBLIOGRAPHY





zur Hausen H, Meinhof W, Scheiber W, Bornkamm GW (1974). Attempts to detect
virus specific ADN in human tumors. I. Nucleic acid hybridizations with complementary
RNA of human wart virus. Int J Cancer 13: 650-6.
Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV et al (1999).
Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J
Pathol 189: 12-9.
zur Hausen H (1996). Papillomavirus infections-a major cause of human cancers.
Biochim Biophys Acta 1288: F55-78.
zur Hausen H (2002). Papillomaviruses and cancer: from basic studies to clinical
application. Nat Rev Cancer 2: 342-50.
Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ
(2003). Epidemiologic classification of human papillomavirus types associated with
cervical cancer. N Engl J Med 348 (6): 518-27.
MASTER DIAGNÓSTICA
Avda. Conocimiento 100. P.T. Ciencias de la Salud; 18016-Granada (Spain)
master@vitroweb.com www.masterdiagnostica.com
12
Rev: 2014-09