PreCursor-M AnoGYN Assay

Methylation-specific molecular assay to detect promoter

hypermethylation of the human genes ASCL1 and ZNF582.

Robust. Reliable.

Special Features

• One-step PCR: Multiplex format enabling simultaneous amplification and detection of the two targets
and control in a single reaction
• Ready-to-use mastermix: Just add bisulfite-converted DNA
• Reliable: - Robust1
- Good reproducibility1
- Built-in check for sample quality and normalization.
• For research use only (RUO). Not for use in diagnostic procedures.

Technical Specifications

• Method: Multiplex real-time methylation-specific PCR assay

• Target: Human genes ASCL1 and ZNF582
• Built-in reference and sample quality control: β-actin (ACTB)
• Calibrator: Contains a standard concentration of low-copy DNA fragments of the two targets ASCL1,
ZNF582 and the control gene ACTB – also used for run validation
• Sample types: Bisulfite-converted DNA prepared from human samples, for instance formalin-fixed paraffin
embedded anal biopsies or anal swab specimens
• Sample volume: 5 µL of bisulfite-converted DNA
• Total time to result: Ranging between 2 to 24 hours depending on the level of automation (incl. DNA
extraction and bisulfite conversion)
• Equipment: Mic qPCR cycler (Bio Molecular Systems), Rotor-Gene Q 5plex system (Qiagen) or ViiA 7 qPCR
system (Thermo Fisher Scientific)1
 Workflow

Bisulfite-conversion with prior DNA extraction

and DNA quality control
Swab Tube Sample Thaw

1. Perform standard DNA extraction

Vortex Centrifuge Incubator Spin

Swab Tube Sample Thaw

C U
2. Measure DNA concentration
C U
Discard Urine sample Pipette Cool down

C U

Vortex
3. Aliquot DNA eluate. DNA Centrifuge Incubator Spin

Prepare Add Master mix on ice Transfer

concentration input for bisulfite-
conversion can range from 100 ng
to 2 µg to obtain valid results

Load Keep on ice C U

Isothermal amplification Falcon Tube
Discard Urine sample Pipette Cool down
Swab Swab Tube
4. Bisulfite-conversion with EZ DNA
Tube Sample Sample Thaw Thaw

C U Methylation Kit or EZ DNA Methylation

Lightning Kit according to the
manufacturer’s recommendation

Tube Sample Swab Tube

Thaw Prepare Add Sample Master Thaw
mix on ice Transfer
Vortex Vortex Centrifuge Centrifuge Incubator Incubator Spin Spin

Thaw
qPCR Reaction Results
Centrifuge Incubator Vortex Spin Load Centrifuge Keep on ice
Incubator Isothermal
Spin amplification Falcon Tube
Discard Discard Urine sample Urine sample Pipette Pipette Cool down Cool down

Amplification
12

10

Normalised Fluorescence
8

90 min.
6

1. Thaw theDiscard 2. Briefly 3. Short spin 4. Dispense 5. Add 5 μl 6. Place tubes in the Mic qPCR
4

Urine sample Pipette Cool down Urine sample Pipette Cool down
2
Add Add Master mix on ice Master mix on ice
PreCursor-M vortex 15μl of of sample cycler and run amplification
Prepare Prepare Transfer Transfer
Spin
0

AnoGYN assay masterCmix U /controls protocol (90 minutes)

0 5 10 15 20 25 30 35 40
Cycles

reagents ZNF582
ACTB
ASCL1

Add Master mix on ice

Prepare Add Master mix on ice Transfer
Load Load Keep on ice Transfer Keep on ice Isothermal amplification Isothermal amplification
Falcon Tube Falcon Tube

Cool down

Order Information
Keep on ice Load
Isothermal amplification Falcon Tube Keep on ice Isothermal amplification Falcon Tube

Description Approval Package size Catalogue number

PreCursor-M AnoGYN
Transfer RUO 72 tests 81524

Reference paper
cation Falcon Tube

1. Rozemeijer K. et al. Tumour Virus Res. 2023 Dec 30;17:200275.

© Fujirebio Europe N.V., February 2024, FRE-316

Distributed by Fujirebio Europe N.V.

Technologiepark 6 – 9052 Gent – Belgium
Tel. +32 (0)9 329 13 29 – Fax. +32 (0)9 329 19 11
PreCursor-M AnoGYN test is a registered trademark of Self-screen B.V. www.fujirebio.com