MTHFR A1298C Q - PCR Alert kit - ELITech Group · Product «MTHFR A1298C Q - PCR Alert Kit», ref. RTSD14, can be used in association with the ... Another specific probe for the mutated

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MTHFR A1298C Q - PCR Alert kit Ref. RTSD14

Composto da: Composed by: Composé par:

Compuesto por: Composta por:

Komponiert von:

MTHFR A1298C Q - PCR Alert AmpliMIX RTSD14-M MTHFR A1298C Q - PCR Alert AmpliPROBE RTSD14-P Q - PCR Alert AmpliMASTER RTS000

ELITechGroup S.p.A. C.so Svizzera, 185

10149 Torino ITALY Offices: Tel. +39-011 976 191 Fax +39-011 936 76 11

E. mail: [email protected] WEB site: www.elitechgroup.com

Notice of Change nr. SCH mRTSD14_05_en dated 28/10/13

NOTICE of CHANGE dated 28/10/13

IMPORTANT COMMUNICATION FOR THE USERS OF PRODUCT:

«MTHFR A1298C Q - PCR Alert Kit » Ref. RTSD14

In the Instructions for Use Manual (IFU), SCH mRTSD14, some changes were introduced regarding:

Product «MTHFR A1298C Q - PCR Alert Kit», ref. RTSD14, can be used in association with the «ELITe STAR System» (ELITechGroup S.p.A.) for the automatic extraction of nucleic acids.

Detailed instructions are reported in the enclosed Instructions for Use manual (IFU).

The assay analytical principle and test reagents ar e unchanged.

PLEASE NOTE

LA REVISIONE DI QUESTO IFU E’ COMPATIBILE ANCHE CON LA VERSIONE PRECEDENTE DEL KIT (vedi Package Insert incluso nel kit)

THE REVIEW OF THIS IFU IS ALSO COMPATIBLE WITH THE PREVIOUS VERSION OF THE KIT (see Package Insert included in the kit)

CET IFU MIS A JOUR ANNULE ET REMPLACE ET EST PARFAI TEMENT COMPATIBLE AVEC LA VERSION PRECEDENTE DU KIT (voir Package Insert inclus dans le kit)

LA REVISIÓN DE ESTE IFU ES COMPATIBLE TAMBIÉN CON L A VERSIÓN ANTERIOR DEL KIT (véa Package Insert incluido en el kit)

A REVISÃO DO ESTE IFU ÉTAMBÉM COMPATÍVEL COM A VERS ÃO ANTERIOR DO KIT (ver Package Insert incluído no kit)

DIE REVIEW VON DIESER IFU IST KOMPATIBLE MIT DER VO RIGE VERSION VON DEM KIT (Sehen Sie Package Insert im Kit enthalten)


10149 Torino ITALY Offices: Tel. +39-011 976 191 Fax +39-011 936 76 1 1


SCH mRTSD14M_en 28/10/13 Review 05 Page 1/15

MTHFR A1298C Q - PCR Alert AmpliMIX allele determination of MTHFR A1298C SNP

TABLE OF CONTENTS INTENDED USE page 1 ASSAY PRINCIPLE page 2 PRODUCT DESCRIPTION page 2 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 OTHER PRODUCTS REQUIRED page 3 WARNINGS AND PRECAUTIONS page 3 SAMPLES AND CONTROLS page 5 PROCEDURE page 6 PROCEDURE LIMITATIONS page 10 PERFORMANCE CHARACTERISTICS page 11 REFERENCES page 13 TROUBLESHOOTING page 14 SYMBOLS page 15

INTENDED USE «MTHFR A1298C Q - PCR Alert AmpliMIX» is part of a qualitative amplification assay of nucleic acids for allele determination of the locus of 5,10-methylene tetrahydrofolate reductase (MTHFR) for single nucleotide polymorphism (SNP) A1298C (E429A) in DNA samples extracted from whole blood collected in EDTA.

The product is intended for use, alongside clinical data and other laboratory tests, in the assessment of risk of deep vein thrombosis.

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SCH mRTSD14M_en 28/10/13 Review 05 page 2/15

ASSAY PRINCIPLE

The assay involves a real-time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real-time).

In each well, an amplification reaction is carried out specific for the region of the human gene that codifies MTHFR affected by SNP A1298C using the DNA extracted from the samples being tested. A specific probe for the normal MTHFR 1298A allele (E429) labelled with FAM fluorophore is activated when hybridized with the “normal” product of the amplification reaction. Another specific probe for the mutated allele MTHFR 1298C (A429) labelled with VIC fluorophore is activated when hybridized with the “mutated” product of the amplification reaction. Fluorescence emission increases as the specific products of the amplification reaction increase and is measured and recorded by the instrument. The processing of the data determines which alleles are present in the DNA extracted from the starting sample.

System standardization was carried out on Applied Biosystems 7000 series instruments.

PRODUCT DESCRIPTION The product supplies the mixture of AmpliMIX primer oligonucleotides for real-time amplification in a stabilizing solution, pre-dosed in aliquots into four disposable test tu bes . Each test tube contains 110 µL of solution, sufficient for 24 tests.

The primer oligonucleotides are specific for the region of the human MTHFR gene affected by SNP A1298C.

The product provides 96 determinations , including controls.

MATERIALS PROVIDED IN THE PRODUCT

Component Description Quantity Composition Labelling

MTHFR A1298C AmpliMIX primer oligonucleotides mixture 4 x 110 µL

Oligonucleotides, TRIS base, TRIS hydrochloride,

Glycerol, Triton X-100 -

MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex powder-free gloves or similar material. - Vortex mixer. - Bench microcentrifuge (12,000 - 14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement (0.5-10 µL, 2-20 µL, 5-50 µL,

50-200 µL, 200-1000 µL). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system.


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OTHER PRODUCTS REQUIRED

The reagents for DNA extraction from the test samples, the reagents optimized for amplification, the detection reagents (fluorescent probes) and heterozygous control DNA are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended:

For manual nucleic acid extraction, it is recommended the use of generic products «EXTRAcell» (ELITechGroup S.p.A., code EXTD02), kit for DNA extraction from cellular samples; the kit enables 50 extractions.

For automatic nucleic acid extraction, it is recommended the use of generic product by ELITechGroup S.p.A. «ELITe STAR 200 Extraction kit» (ELITechGroup S.p.A., code INT011EX), kit for extraction of DNA and RNA from non-cellular and cellular samples with «ELITe STAR instrument» (ELITechGroup S.p.A., code INT010), hereafter indicated as «ELITe STAR» .

«ELITe STAR 200 Extraction Kit» (ELITechGroup S.p.A., code INT011EX) and «ELITe STAR Instrument» (ELITechGroup S.p.A., code INT010) constitute «ELITe STAR System» .

«Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification and allele determination; the product provides 96 reactions.

«MTHFR A1298C Q - PCR Alert AmpliPROBE» (code RTSD14-P), fluorescent probes for allele determination; the product provides 96 reactions.

«MTHFR A1298C - Positive Control» (code CTRD14), heterozygous control of plasmid DNA; the product provides 25 reactions.

When a 7500 Fast Dx Real-Time PCR Instrument is used, it is recommended the use of generic product: «Q - PCR Microplates Fast» (ELITechGroup S.p.A., code RTSACC02), microplates with 0.1 mL wells and adhesive sealing sheets for real time amplification.

WARNINGS AND PRECAUTIONS This product is exclusively for in vitro use.

Warnings and general precautions

Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121°C for one hour before disposal.

Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal.

Wear suitable protective clothing and gloves and protect eyes / face.

Never pipette solutions by mouth.

Do not eat, drink, smoke or apply cosmetic products in the work areas.

Wash hands carefully after handling samples and reagents.

Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the product before running the assay.

Follow the instructions provided with the product while running the assay.

Do not use the product after the expiry date.

Only use the reagents provided in the product and those recommended by the manufacturer.

Do not mix reagents from different batches.


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Warnings and precautions for molecular biology

Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products.

It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never introduce an amplification product in the area designed for extraction / preparation of amplification reactions.

It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification / detection of amplification products to the area designed for the extraction / preparation of the amplification reactions.

The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Test tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA.

Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA.

Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose.

Warnings and precautions specific to components

The test tubes containing AmpliMIX are disposable and therefore must be used once only in the preparation of the reaction mixture.

AmpliMIX carries the following safety warnings (S):

S 23-25. Do not breathe gas/fumes/vapour/spray. Avoid contact with eyes.


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SAMPLES AND CONTROLS

Samples

This product must be used with DNA extracted from the following biological samples: whole blood collected in EDTA

The whole blood samples to be used for DNA extraction must be collected in EDTA according to laboratory guidelines, transported at +2° / +8°C an d stored at +2° / +8°C for a maximum of three days, otherwise they must be frozen and stored at -20°C f or a maximum of thirty days. It is advisable to split the samples that are to be stored frozen into aliquots in order to prevent repeated cycles of freezing and thawing.

N.B.: When the manual nucleic acid extraction is carried out using «EXTRAcell» kit, please, carefully follow the instructions for use manual for pre-treatment of clinical samples.

N.B.: When the nucleic acid extraction is carried out from whole blood sample with ELITe STAR System and software version 3.4.13 please use the extraction protocol UUNI_E200S200_ELI and follow these directions: ELITe STAR is able to use a sample primary tube, the sample volume required for the extraction is 200 µL . The samples in primary tubes can be loaded directly on the ELITe STAR . It is always requested a minimum dead volume of 400 - 600 µL depending on sample primary tube type. Further information and details on primary tubes and the extraction procedure can be found in the instruction for use of extraction kit.

It is recommended to release approx. 175 ng of DNA extract into the amplification reaction (equivalent to approx. 25,000 cells). Do not release into the amplification reaction a hi gher quantity of DNA extract than 200 ng , in order to prevent the problem of non-specific hybridization or inhibition of the fluorescence emission.

Interfering substances

The DNA extracted from the starting sample must not contain heparin in order to prevent the problem of inhibition and the possibility of frequent invalid results. The DNA extracted from the starting sample must not contain haemoglobin in order to prevent the problem of inhibition of the amplification and fluorescence emission and the possibility of frequent invalid, undetermined or incorrect results. There are no data available concerning the problem of inhibition caused by drugs.

Amplification controls

It is absolutely mandatory to validate each amplification session with a positive control reaction and a negative control reaction. For the negative control, use sterile bidistilled water (not supplied with product) added to the reaction in place of the DNA extracted from the sample. For the positive control, use the DNA extracted from a heterozygous sample that has already tested positive, or «MTHFR A1298C - Positive Control» .

Quality controls

It is recommended to validate the whole analysis procedure of each extraction and amplification session by processing a normal sample, a mutated homozygous sample and a heterozygous sample that have already been tested with a different method and verify the obtained results were in accordance to this method.


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PROCEDURE

Setting up the real-time amplification session (To be performed in the amplification / detection area of the amplification products)

Before starting the session it is important to do the following: - referring to the instrument documentation, switch on the real-time thermal cycler, switch on the control computer, launch the special software and open an "absolute quantification" session; - referring to the instrument documentation, first set the "detector" for the "nor MTHFR 1298A" normal allele probe with the reporter as "FAM" and the "quencher" as "none" (NFQ = non-fluorescent quencher); - referring to the instrument documentation, secondly set the "detector" for the "mu MTHFR 1298C" mutated allele probe with the reporter as "VIC" and the "quencher" as "none" (NFQ = non-fluorescent quencher); - referring to the instrument documentation, for each well used in the microplate, set the "detector" (type of fluorescence that is to be measured), the "passive reference" as “ROX” (normalisation of measured fluorescence) and the type of reaction (sample, negative amplification control, positive amplification control). Add this information to the Work Sheet enclosed at the end of this instruction manual or print the microplate organisation. The Work Sheet must be followed carefully during the transfer of the reaction mixture and samples into the wells.

Note: Below is an example of how the analysis of 6 samples can be organized.

S1

S2

S3

S4

S5

S2

C

PC

Key: S1 - S6: Samples to be analysed; NC: Negative amplification control; PC: Positive amplification control. Referring to the instrument documentation, set the parameters of the thermal cycle on the thermal cycler, setting 30 cycles and a reaction volume of 25 µL .

When a 7300 Real-Time PCR System instrument is used, set “Run mode: standard 7300”.

When a 7500 Fast Dx Real-Time PCR Instrument is used, set “Run mode: fast 7500”.

Amplification thermal cycle Phase Temperature Timing

Decontamination 50° C 2 min.

Initial denaturation 95° C 10 min.

30 cycles

95° C 15 sec. 60° C

(fluorescence acquisition)

1 min.

- save the settings of the "absolute quantification" session using an unambiguous and easy-to-recognise label (e.g. "year-month-day- MTHFR A1298C").


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Preparation of the real-time amplification (To be performed in the extraction / preparation area of the amplification reaction)

Before starting the session it is important to do the following: - remove and thaw the test tubes containing the samples to be analysed. Centrifuge the tubes to

bring the contents to the bottom and keep in ice; - remove and thaw the AmpliMIX test tubes needed for the session, remembering that the contents

of each tube are sufficient for 24 reactions . Centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice;

- remove and thaw the same number of test tubes of AmpliPROBE as the AmpliMIX tubes. Centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice;

- remove and thaw the same number of test tubes of AmpliMASTER as the AmpliMIX tubes. Write "MTHFR A1298C" and the date on the test tube label using indelible ink. Centrifuge the tubes for 5 seconds to bring the contents to the bottom and keep in ice;

- remove and thaw the Positive Control test tubes. Centrifuge the tube for 5 seconds to bring the contents to the bottom and keep in ice;

- if necessary, cut the Amplification microplate to separate the part that will be used in the session, being careful to handle it with powder-free gloves and not to damage the wells.

1. Transfer 100 µL of AmpliMIX to the AmpliMASTER tubes. Mix well and pipette the volume of 100 µL three times into the mix.

2. Transfer 100 µL of AmpliPROBE to the AmpliMASTER tube. Mix well and pipette the volume of 100 µL three times into the mix.

3. Vortex on a low setting for 5 seconds, avoiding the creation of foam.

4. Centrifuge the tubes for 5 seconds to bring the contents to the bottom. 5. Carefully deposit 20 µL of the reaction mixture obtained in this way on the bottom of the Amplification

microplate wells, as previously established on the Work Sheet.

N.B.: If not all the reaction mixture is used, store the remaining volume in the dark at -20°C for a maximu m of one month in the test tube labelled "MTHFR A1298C" . Freeze and thaw the reaction mixture only once .

6. Carefully deposit 5 µL of DNA extracted from the first sample in the reaction mixture in the corresponding well of the Amplification microplate , as previously established on the Work Sheet . Proceed in this way for all the other DNA extracts .

7. Carefully deposit 5 µL of sterile bidistilled water (not supplied with the product) in the reaction mixture in the well of the negative control Amplification microplate , as previously established on the Work Sheet .

8. Carefully deposit 5 µL of Positive Control in the reaction mixture in the Amplification microplate well of the amplification positive control, as previously established on the Work Sheet .

9. Carefully seal the Amplification microplate using the Amplification Sealing Sheet .

10. Transfer the Amplification microplate to the real-time thermal cycler in the amplification/detection area for amplification products and start the amplification thermal cycle.

N.B.: At the end of the thermal cycle the Amplification microplate with the reaction products must be removed from the instrument and eliminated without producing environmental contaminations. In order to avoid the spilling of the reaction products, the Amplification Sealing Sheet must not to be removed from the Amplification microplate .


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Interpreting the results

The values of FAM fluorescence emitted by the specific probe for the MTHFR 1298A normal allele are identified by the detector "nor MTHFR 1298A" (FAM). The values of VIC fluorescence emitted by the specific probe for the MTHFR 1298C mutated allele are identified by the detector "mu MTHFR 1298C" (VIC). These fluorescence values in the amplification reactions must be analysed by the software of the instruments.

Before analysing, referring to the instrument documentation, it is necessary to: - manually set the calculation range for the fluorescence back ground level (Baseline) from cycle

6 to cycle 15;

When a 7300 Real-Time PCR System instrument is used:

- set manually the Threshold for the FAM detector “nor MTHFR 1298A” to 0.2; - set manually the Threshold for the VIC detector "mu MTHFR 1298C" to 0.2.

When a 7500 Fast Dx Real-Time PCR Instrument is used:

- set manually the Threshold for the FAM detector “nor MTHFR 1298A” to 0.1; - set manually the Threshold for the VIC detector "mu MTHFR 1298C" to 0.1.

The values of fluorescence emitted by the specific probes for the MTHFR 1298A (FAM) normal allele and the MTHFR 1298C (VIC) mutated allele in the amplification reactions and the Threshold value of fluorescence are used to determine the Threshold cycle (Ct), the amplification cycle in which the value of fluorescence emitted by each probe has reached the Threshold level.

The Ct values for the specific probes for the normal allele and the mutated allele in the Negative control amplification reaction are used to validate amplification and detection as shown in the following table:

Negative control amplification Amplification / Detection

Ct FAM = Undetermined CORRECT

Ct VIC = Undetermined CORRECT

Where Ct FAM is the Threshold cycle of the normal allele (FAM "nor MTHFR 1298A") and Ct VIC is the Threshold cycle of the mutated allele (VIC "mu MTHFR 1298C").

If the result of the Negative control amplification reaction is other than Ct Undetermined , the target DNA has been detected in the amplification reaction. Problems have occurred during the amplification phase (contamination) which may cause incorrect results. The session is invalid and must be repeated from the amplification phase.

The Ct values for the specific probes for the normal allele and the mutated allele in the positive control amplification reaction are used to validate amplification and detection as shown in the following table:

Positive control amplification Amplification / Detection

20 ≤ Ct FAM or Ct VIC ≤ 25 CORRECT

0 ≤ |Ct FAM - Ct VIC| ≤ 1.5 CORRECT

Where |Ct FAM - Ct VIC| is the absolute value of the difference between the Threshold cycle of the normal allele (FAM "nor MTHFR 1298A") and the Threshold cycle of the mutated allele (VIC "mu MTHFR 1298C").

If the result of the amplification reaction of the Positive Control is not within the limits, the target DNA has not been detected correctly in the amplification reaction. Problems have occurred during the amplification or detection phase (incorrect preparation of the reaction mixture, incorrect dispensing of the reaction mixture or the positive control, probe or positive control degradation, incorrect setting position of the positive control, incorrect setting of the thermal cycle) which may cause result errors. The session is invalid and must be repeated from the amplification phase.


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In the amplification reactions of each sample , the Ct values of normal and mutated probe are used to validate amplification and detection and for allele determination of the genotype.

N.B.: Verify with the instrument software (Results > Amplification plot > delta Rn vs Cycle) that the Ct was determined by a fast and regular increase of the fluorescence values and not by peaks or an increase of the background (irregular or high background).

This product is able to detect a minimal quantity of about 50 ng of human genomic DNA per reaction (see Performance Characteristics paragraph, page 11).

The results as Ct of the amplification reactions of each sample are used as described in the following table:

Amplification of the sample Sample

suitability Assay result Genotype of the

sample

Ct FAM > 27.5 and Ct VIC > 27.5 not suitable invalid -

Ct FAM ≤ 27.5

or

Ct VIC ≤ 27.5

Ct FAM Undet. and Ct VIC ≤ 27.5

or

|Ct FAM - Ct VIC| ≥ 2.5 with Ct FAM > Ct VIC

suitable valid MUTATED HOMOZYGOUS

Ct FAM ≤ 27,5 and Ct VIC Undet.

or

|Ct FAM - Ct VIC| ≥ 2.5 with Ct FAM < Ct VIC

suitable valid NORMAL

HOMOZYGOUS

2.0 < |Ct FAM - Ct VIC| < 2.5 not suitable valid UNDETERMINED

0 ≤ |Ct FAM - Ct VIC| ≤ 2.0 suitable valid HETEROZYGOTE

If the result of the amplification reaction of a sample is: Ct FAM > 27.5 and Ct VIC > 27.5 , this means that problems have occurred during the amplification phase (inefficient or invalid amplification) or in the extraction phase (absence of DNA or presence of inhibitors or insufficient number of cells in the starting sample) which may cause incorrect results. The sample is not suitable, the assay is invalid and must be repeated beginning with extraction of a new sample.

If the result of the amplification reaction of a sample is: absolute value of the difference between Ct FAM and Ct VIC from 2.0 to 2.5 , this means that problems have occurred during amplification (inefficient amplification) or extraction (presence of inhibitors or excess number of cells in the starting sample) preventing allele determination of the genotype. The sample is not suitable, the assay is undetermined and must be repeated beginning with extraction of a new sample.

The results obtained with this assay must be interpreted in consideration of all the clinical data and the other laboratory tests done on the patient.


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PROCEDURE LIMITATIONS

Use only DNA extracted from the following human samples with this product: whole blood collected in EDTA.

Do not use a higher quantity of DNA extract with this product than the recommended amount: higher quantities of DNA extract could lead to incorrect or undetermined results.

Do not use DNA extracted from heparinized samples with this product: heparin inhibits the amplification reaction of nucleic acids and causes invalid results.

Do not use DNA extract that is contaminated with haemoglobin with this product: haemoglobin inhibits the amplification reaction of nucleic acids and may cause invalid results; haemoglobin inhibits the fluorescence emission and could cause undetermined or incorrect results.

There are no data available concerning the problem of inhibition caused by drugs.

The results obtained with this product are subject to the correct collection, transport, storage and preparation of samples. To avoid result errors it is therefore necessary to take particular care during these phases and to carefully follow the instructions provided with the products for nucleic acid extraction.

Owing to its high analytical sensitivity, the real-time amplification assay of nucleic acids used in this product is subject to contamination from clinical samples, positive controls and the amplification reaction products themselves. Contamination leads to incorrect results. The product has been designed in such a way as to reduce contamination; nevertheless, this phenomenon can only be prevented by following good laboratory practices and by complying scrupulously with the instructions provided in this manual.

This product must be handled by personnel trained in the processing of potentially infective biological samples and chemical preparations classified as dangerous to prevent accidents with potentially serious consequences for the user and other persons.

This product requires the use of work clothes and premises that are suitable for the processing of potentially infective biological samples and chemical preparations classified as dangerous to prevent accidents with potentially serious consequences for the user and other persons.

This product must be handled by personnel trained in molecular biology techniques, such as extraction, amplification and detection of nucleic acids, to avoid result errors.

It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products to prevent incorrect results.

This product requires the use of special clothing and instruments for extraction / preparation of amplification reactions and for amplification / detection of amplification products to avoid incorrect results.

As with any diagnostic device, the results obtained with this product must be interpreted in consideration of all the clinical data and other laboratory tests done on the patient.

As with any diagnostic device, there is a residual risk of obtaining invalid or incorrect results with this product. This residual risk cannot be eliminated or reduced any further. In particular situations, this residual risk can contribute to incorrect decisions with potentially grave consequences for the patient.


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PERFORMANCE CHARACTERISTICS

Analytical sensitivity: detection limit

In terms of the detection limit, the analytical sensitivity of this assay enables identification of approx. 14,000 target DNA molecules (equivalent to the genomes of 7,000 cells or 50 ng of human genomic DNA) in 5 µL of DNA extract added to the amplification reaction.

The detection limit was tested using human genomic DNA whose initial concentration was determined by reading the absorption at 260 nm on the spectrophotometer. 50 ng / reaction of the diluted human genomic DNA was used in 50 repeats for the whole procedure of analysis, amplification and detection with ELITechGroup S.p.A. products (see paragraph on accessory products). All repeats were valid and correctly determined.

Sample No. correct incorrect invalid undetermined

Human genomic DNA 50 50 0 0 0 Analytical sensitivity: ability to identify the mut ated allele

In terms of its capacity to identify the mutated allele, the analytical sensitivity of the assay is higher than 99%.

The capacity to identify the mutated allele (no. of correct results from valid results) was tested using a number of known-genotype normal heterozygous and mutated homozygous whole blood samples. These samples were used for the whole analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products.

Out of 224 determinations 222 were valid (at least one Ct less than or equal to 27.5). Out of 222 valid determinations, 222 gave a correct result, no samples gave an incorrect result (no heterozygotes were mistaken for normal homozygotes and no mutated homozygotes were mistaken for heterozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5).

Samples No. correct incorrect invalid undetermined

Heterozygotes 112 111 0 1 0

Mutated homozygotes 112 111 0 1 0 Analytical specificity: inability to identify mutat ed allele as normal

In terms of inability to identify the normal allele as mutated, the analytical specificity of this assay is greater than 99%.

The capacity to correctly identify the normal allele (no. of correct results from valid results) was tested using a number of known-genotype normal homozygous and heterozygous whole blood samples. These samples were used for the whole analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products.

Out of 224 determinations 223 were valid (at least one Ct less than or equal to 27.5). Out of 223 valid determinations, 223 gave a correct result, no samples gave an incorrect result (no normal homozygotes were mistaken for heterozygotes and no heterozygotes were mistaken for mutated homozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5).


Normal homozygotes 112 112 0 0 0



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Robustness: Ct limit for validation

The robustness of this assay enables a frequency of valid samples of 99.4% with a Ct limit for validation of 27.5.

The Ct limit was established using a number of known-genotype normal homozygous, heterozygous and mutated homozygous whole blood samples. These samples were used for the whole analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products.

Out of 336 determinations, 334 samples were valid (at least one of the Ct less than or equal to 27.5) and 2 samples were invalid (both Ct greater than 27.5).




Mutated homozygotes 112 111 0 1 0 Robustness: difference between homozygote Ct values

The robustness of this assay enables determination of homozygotes at a frequency greater than 99% with a Ct difference between the two alleles that is greater than or equal to 2.5.

The Ct difference between the two alleles for homozygotes was established using a number of known-genotype homozygous and mutated homozygous whole blood samples. These samples were used for the whole analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products.

Out of 224 determinations 223 were valid (at least one Ct less than or equal to 27.5). Out of 223 valid determinations, 223 gave a correct result, no samples gave an incorrect result (no normal homozygotes were mistaken for heterozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5).



Mutated homozygotes 112 111 0 1 0 Robustness: difference between heterozygote Ct valu es

The robustness of this assay enables determination of heterozygotes at a frequency greater than 99% with a Ct difference between the two alleles of 0 to 2.0

The Ct difference between the two alleles for heterozygotes was established using a number of known-genotype heterozygous whole blood samples. These samples were used for the whole analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products.

Out of 112 determinations 111 were valid (at least one Ct less than or equal to 27.5). Out of 111 valid determinations, 111 gave a correct result, no samples gave an incorrect result (no heterozygotes were mistaken for homozygotes), no samples gave undetermined results (Ct difference between 2.0 and 2.5)




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Reproducibility The reproducibility of this assay, in terms of its capacity to obtain the same results from the same samples in different sessions, is greater than 98%.

The ability to obtain the same results from the same samples in different sessions was tested using a number of known-genotype normal homozygous, heterozygous and mutated homozygous whole blood samples. These samples were used for the whole analysis procedure, manual extraction with «EXTRAcell» kit, amplification and detection with ELITechGroup S.p.A. products.

Out of 3 allele determination sessions carried out on the same 77 samples on different days, 77 results were concordant.

Samples No. sessions concordant discordant

Normal homozygotes 28 3 28 0

Heterozygotes 28 3 28 0

Mutated homozygotes 21 3 21 0 Diagnostic specificity The diagnostic specificity of this assay, as the ability to not identify as mutated the normal allele was tested on samples of archival blood from subjects with normal homozygous and known heterozygous genotype.

The ability to correctly detect the normal allele was tested using 26 samples of whole blood with known normal homozygous genotype and 22 samples of known heterozygous genotype from different donors. These samples were used for the whole analysis procedure, automatic extraction with «ELITeSTAR System» , amplification and detection with ELITechGroup S.p.A. products. Out of 48 determinations 47 have given a valid result.

The results are summed up in the following table. Samples N concordant discordant Not valid

Normal homozygous 26 26 0 0

Heterozygous 22 23 0 1

In this test the diagnostic specificity was equal to 100%. Diagnostic sensitivity The diagnostic sensitivity of this assay, as the ability to identify the mutated allele, was tested on samples of archival blood from subjects with known heterozygous genotype.

The ability to correctly detect the mutated allele even in single couple was tested using 23 samples of whole blood with known heterozygous genotype and 12 samples of known homozygous mutated genotype from different donors. from different donors. These samples were used for the whole procedure of analysis, automatic extraction with «ELITeSTAR System» , amplification and detection with ELITechGroup S.p.A. products. Out of 34 determinations 32 have given a valid result, one determination gave a discordant result.

The results are summed up in the following table. Samples N concordant discordant Not valid Heterozygous 22 21 0 1

Homozygous mutated 12 11 1 0

In this test the diagnostic sensitivity was equal to 100%. N.B.: The complete data and results of the tests carried out to evaluate the performance characteristics of the product are recorded in Section 7 of the Product Technical File "MTHFR A1298C Q - PCR Alert AmpliMIX" and "MTHFR A1298C Q - PCR Alert AmpliPROBE", FTP RTSD14.

REFERENCES

I. Weisberg et al. (1998) Mol. Genet Metab.64: 169 - 172


RTSD14-M


TROUBLESHOOTING Target DNA present in the negative control reaction

Possible causes Solutions

Dispensing error on the microplate.

Avoid spilling the contents of the sample test tube. Always change tips between one sample and another. Take care when dispensing samples, negative and positive controls onto the microplate and comply with the work sheet.

Error while setting the instrument Check the position settings of the samples, negative and positive controls on the instrument.

Microplate badly sealed. Take care when sealing the microplate.

Contamination of the sterile bidistilled water. Use a new aliquot of sterile water.

Contamination of the amplification mix. Use a new aliquot of amplification mix.

Contamination of the extraction / preparation area for amplification reactions.

Clean surfaces and instruments with aqueous detergents, wash lab coats, replace test tubes and tips in use.

Amplification absent or not efficient in the Positi ve Control reaction


Error in the preparation of the reaction mixture. Check the volumes of reagent dispensed during preparation of the reaction mixture.

Dispensing error on the microplate.

Take care when dispensing reactions onto the microplate and comply with the work sheet. Check the volumes of reaction mixture dispensed. Check the volumes of positive control dispensed.

Probe degradation. Use a new probe aliquot.

Degradation of the positive control. Use a new aliquot of positive control.

Instrument setting error. Check the position settings of positive control reaction on the instrument. Check the thermal cycle settings on the instrument.

High frequency of invalid or undetermined results i n the sample reactions


DNA extract contaminated by haemoglobin. Repeat the extraction avoiding contamination of the final product with haemoglobin.

Excess DNA extract in the reaction.

Assess the number of leukocytes in the sample from the blood count value; repeat the extraction from 500,000 cells. Do not add more than 200 ng of genomic DNA extract to the reaction.

Problems with the optical system. Check for contamination with the fluorophore of the thermal block or the lens. Check for obstacles in the optical path.


RTSD14-M


SYMBOLS

Catalogue number. Upper temperature limit. Batch code. Use by (last day of month). In vitro diagnostic medical device. In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic

medical devices. Contents sufficient for "N" tests. Contents. Please refer to the instructions for use. Manufacturer. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or "Q - PCR Standard". No general patent or other license of any kind other then this specific right of use from purchase is granted hereby.

CONT

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SCH mRTSD14P_en 28/10/13 Review 05 Page 1/4

MTHFR A1298C Q - PCR Alert AmpliPROBE

allele determination of MTHFR A1298C SNP

TABLE OF CONTENTS INTENDED USE page 1 PRODUCT DESCRIPTION page 1 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 OTHER PRODUCTS REQUIRED page 2 WARNINGS AND PRECAUTIONS page 2 PROCEDURE page 4 REFERENCES page 4 SYMBOLS page 4

INTENDED USE «MTHFR A1298C Q - PCR Alert AmpliPROBE» is part of a qualitative amplification assay of nucleic acids for allele determination of the locus of 5,10-methy lenetetrahydrofolate reductase (MTHFR) for single nucleotide polymorphism (SNP) A1 298C (E429A) in DNA samples extracted from whole blood collected in EDTA.

.The product is intended for use, alongside clinical data and other laboratory tests, in the assessment of risk of deep vein thrombosis.

PRODUCT DESCRIPTION The product supplies the mixture of AmpliPROBE fluorescent probes for hybridization in a stabilizing solution, pre-dosed in aliquots into four disposable test tub es. Each test tube contains 110 µL of solution, sufficient for 24 tests.

The normal allele probe, labelled with FAM fluorophore and blocked by the MGB-NFQ group, is specific for the normal 1298A (E429) region of the human gene codifying MTHFR.

The normal allele probe, labelled with VIC fluorophore and blocked by the MGB-NFQ group, is specific for the mutated 1298C (A429) region of the human gene codifying MTHFR.

The product provides 96 determinations , including controls.

-20°C RTSD14-P




MTHFR A1298C Q - PCR Alert AmpliPROBE allele determination of MTHFR A1298C SNP

RTSD14-P


MATERIALS PROVIDED IN THE PRODUCT


MTHFR A1298C AmpliPROBE mixture of fluorescent probes labelled with FAM / MGB-NFQ

and VIC / MGB-NFQ 4 x 110 µL

fluorescent oligonucleotides, TRIS base, TRIS hydrochloride,

Glycerol, Triton X-100 -

MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT - Laminar airflow hood. - Disposable latex powder-free gloves or similar material. - Vortex mixer. - Bench microcentrifuge (12,000 - 14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement (0.5-10 µL, 2-20 µL, 5-50 µL,

50-200 µL, 200-1000 µL). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system.

OTHER PRODUCTS REQUIRED The reagents for DNA extraction from the test samples, the reagents optimized for amplification, the primer reagents (oligonucleotides) and the heterozygous control DNA are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended:

For manual nucleic acid extraction, it is recommended the use of generic products «EXTRAcell» (ELITechGroup S.p.A., code EXTD02), kit for DNA extraction from cellular samples; the kit enables 50 extractions.

For automatic nucleic acid extraction, it is recommended the use of generic product by ELITechGroup S.p.A. «ELITe STAR 200 Extraction kit» (ELITechGroup S.p.A., code INT011EX), kit for extraction of DNA and RNA from non-cellular and cellular samples with «ELITe STAR instrument» (ELITechGroup S.p.A., code INT010), hereafter indicated as «ELITe STAR» .

«ELITe STAR 200 Extraction Kit» (ELITechGroup S.p.A., code INT011EX) and «ELITe STAR Instrument» (ELITechGroup S.p.A., code INT010) constitute «ELITe STAR System» .

«Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates and adhesive sheets for real time amplification and allele determination; the product provides 96 reactions.

«MTHFR A1298C Q - PCR Alert AmpliMIX» (code RTSD14-M), primer oligonucleotides for allele determination; the product provides 96 reactions.

«MTHFR A1298C - Positive Control» (code CTRD14), heterozygous control of plasmid DNA; the product provides 25 reactions.

WARNINGS AND PRECAUTIONS This product is exclusively for in vitro use.


Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121°C for one hour before disposal.


RTSD14-P


Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal.

Wear suitable protective clothing and gloves and protect eyes / face.

Never pipette solutions by mouth.

Do not eat, drink, smoke or apply cosmetic products in the work areas.

Wash hands carefully after handling samples and reagents.

Dispose of leftover reagents and waste in compliance with regulations in force.

Read all the instructions provided with the product before running the assay.

Follow the instructions provided with the product while running the assay.

Do not use the product after the expiry date.

Only use the reagents provided in the product and those recommended by the manufacturer.

Do not mix reagents from different batches.

Do not use reagents from other manufacturers' products.


Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products.

It is necessary to have separate areas for the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never introduce an amplification product in the area designed for extraction / preparation of amplification reactions.

It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction / preparation of amplification reactions and for the amplification / detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification / detection of amplification products to the area designed for the extraction / preparation of the amplification reactions.

The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Test tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA.

Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA.

Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose.

Warnings and precautions specific to reagents

The test tubes containing AmpliPROBE are disposable and therefore must be used once only in the preparation of the reaction mixture.

The AmpliPROBE carries the following safety warnings (S):



RTSD14-P


PROCEDURE

The «MTHFR A1298C Q - PCR Alert AmpliPROBE» product must be used with «Q - PCR Alert AmpliMASTER» and «MTHFR A1298C Q - PCR Alert AmpliMIX» products to obtain the reaction mixture.

AmpliPROBE is ready for use, hence must be added directly to the reaction mixture. The complete procedure involves preparation and execution of an amplification reaction and hybridization with fluorescent probes on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time) and is described in detail in the instructions manual enclosed with the «MTHFR A1298C Q - PCR Alert AmpliMIX» kit.

The performance characteristics and procedure limitations of the complete assay for allele determination of MTHFR A1298C SNP are described in detail in the instructions manual enclosed with the «MTHFR A1298C Q - PCR Alert AmpliMIX» kit.

REFERENCES

I. Weisberg et al. (1998) Mol. Genet Metab.64: 169 - 172

SYMBOLS Catalogue number. Upper temperature limit. Batch code. Use by (last day of month). In vitro diagnostic medical device. In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic

medical devices. Contents sufficient for "N" tests. Contents. Please refer to the instructions for use. Manufacturer. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or "Q - PCR Standard". No general patent or other license of any kind other then this specific right of use from purchase is granted hereby.

CONT

SCH mRTS000_en 28/05/13 Review 04 Page 1/4

Q - PCR Alert AmpliMASTER reagents optimized for real time amplification

TABLE OF CONTENTS INTENDED USE page 1 PRODUCT DESCRIPTION page 1 MATERIALS PROVIDED IN THE PRODUCT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT page 2 ACCESSORY PRODUCTS page 2 WARNINGS AND PRECAUTIONS page 3 PROCEDURE page 4 SYMBOLS page 4

INTENDED USE

«Q - PCR Alert AmpliMASTER» is part of the quantitative amplification assay of nucleic acids for the detection and dosing of a target DNA with the real time amplification method in samples of DNA extract or cDNA obtained from RNA extract and for allele determination with real time amplification method in samples of DNA extract. The product is an accessory of the products in the «Q - PCR Alert AmpliMIX» , «Q - PCR Alert AmpliPROBE» , «Q - PCR Standard» lines and in the «Positive Control» lines by ELITechGroup S.p.A.

The product is intended for use in the preparation of diagnostic assays based on the real time amplification method.

PRODUCT DESCRIPTION

In the complete size (RTS000) the product provides the mixture of reagents optimized for AmpliMASTER real time amplification in a stabilizing solution divided into aliquots in four ready-to-use test tubes, three amplification microplates with 96 wells and three adhesive sheets for amplification . In the reduced size (RTS000/2) the product provides the mixture of reagents optimized for AmpliMASTER real time amplification in a stabilizing solution divided into aliquots in two ready-to-use test tubes, two amplification microplates with 96 wells and two adhesive sheets for amplification .

The reagent mixture provides the buffer system, magnesium chloride, triphosphate nucleotides, ROX fluorophore as the passive reference for the normalisation of the fluorescence, the enzyme Uracil N-glycosidase (UNG) to inactivate contamination from the amplification product, the enzyme Taq DNA polymerase for hot start.

N.B.: the amplification microplates shouldn't be used with the Applied Biosystems 7000 series instruments, provided with "FAST" thermal block.

RTS000 RTS000/2

+8° C

+2° C

ELITechGroup S.p .A. C.so Svizzera, 185


E. mail: [email protected] WEB site : www.elitechgroup.com


RTS000 RTS000/2


In the complete size (RTS000) the product provides 96 determinations , including standards and controls. In the reduced size (RTS000/2) the product provides 48 determinations , including standards and controls.

MATERIALS PROVIDED IN THE PRODUCT Complete size product, code RTS000


AmpliMASTER optimized reagent mixture 4 x 340 µL

TRIS base, TRIS hydrochloride, Glycerol, magnesium chloride,

Deoxyribonucleotide triphosphates, ROX, Uracil-N-glycosidase, Taq

DNA polymerase

-

Amplification microplate microplate with 96 x 0.2 ml wells 3 - -

Amplification Sealing Sheet adhesive sealing sheet 3 - -

Reduced size product, code RTS000/2


AmpliMASTER optimized reagent mixture 2 x 340 µL

TRIS base, TRIS hydrochloride, Glycerol, magnesium chloride,

Deoxyribonucleotide triphosphates, ROX, Uracil-N-glycosidase, Taq

DNA polymerase

-

Amplification microplate microplate with 96 x 0.2 ml wells 2 - -

Amplification Sealing Sheet adhesive sealing sheet 2 - -

MATERIALS REQUIRED BUT NOT PROVIDED IN THE PRODUCT

- Laminar airflow hood. - Disposable latex powder-free gloves or similar material. - Vortex mixer - Bench microcentrifuge (12,000 - 14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement (0.5-10 µL, 2-20 µL, 5-50 µL,

50-200 µL, 200-1000 µL). - Sterile bidistilled water. - Programmable heater with optical fluorescence detection system (thermal cycler for real time).

ACCESSORY PRODUCTS The primer reagents (oligonucleotides), the detection reagents (fluorescent probes), the known-quantity DNA standard and the positive controls on the plasmid DNA, are not included in this product. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended:

«Q - PCR Alert AmpliMIX» series, primer oligonucleotides for real time amplification.

«Q - PCR Alert AmpliPROBE» series, fluorescent probes for real time amplification.

«Q - PCR Standard» series, known-quantity plasmid DNA to obtain the standard curve.

«Positive Control» series, positive control of plasmid DNA.


RTS000 RTS000/2


WARNINGS AND PRECAUTIONS

This product is exclusively for in vitro use.


Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121°C for one hour before disposal. Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. Wear suitable protective clothing and gloves and protect eyes / face. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic products in the work areas. Wash hands carefully after handling samples and reagents. Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the product before running the assay. Follow the instructions provided with the product while running the assay. Do not use the product after the expiry date. Only use the reagents provided in the product and those recommended by the manufacturer. Do not mix reagents from different batches. Do not use reagents from other manufacturers' products.


Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products. It is necessary to have separate areas for the extraction/preparation of amplification reactions and for the amplification/detection of amplification products. Never introduce an amplification product in the area designed for extraction/preparation of amplification reactions. It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction/preparation of amplification reactions and for the amplification/detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification/detection of amplification products to the area designed for the extraction/preparation of the amplification reactions. The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose.

Warnings and precautions specific to components

AmpliMASTER does not carry risk phrases (R) and it carries the following safety warnings (S):



RTS000 RTS000/2


PROCEDURE

The «Q - PCR Alert AmpliMASTER» product must be used with the products in the «Q - PCR Alert AmpliMIX» series and the «Q - PCR Alert AmpliPROBE» line to obtain the reaction mixture.

AmpliMASTER is ready for use, hence must be used directly in the preparation of the reaction mixture.

The complete procedure involves preparation and execution of a real time amplification reaction on a microplate with programmable heater with optical fluorescence detection system (thermal cycler for real time) and is described in detail in the instruction manual enclosed with the products in the «Q - PCR Alert AmpliMIX» series.

The performance characteristics and procedure limitations of the assay including detection and dosing of the target DNA are described in detail in the instruction manual enclosed with the products in the «Q - PCR Alert AmpliMIX» series.

SYMBOLS Catalogue number. Temperature limits. Batch code. Use by (last day of month). In vitro diagnostic medical device. In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic

medical devices. Contents sufficient for "N" tests. Contents. Please refer to the instructions for use. Manufacturer. The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" or “Q - PCR Standard”. No general patent or other license of any kind other then this specific right of use from purchase is granted hereby.

CONT

Documents

MTHFR A1298C Q - PCR Alert kit - ELITech Group · Product «MTHFR A1298C Q - PCR Alert Kit», ref. RTSD14, can be used in association with the ... Another specific probe for the mutated