Package insert

Nanogen Advanced Diagnostics S.r.L. 

Corso Torino, 89/d 

10090 Buttigliera Alta (TO) ITALY 

Offices: 

Tel. +39-011 976 19 1 

Fax +39-011 936 76 11 

E. mail: techsupport@nanogenad.com 

Q - PCR Alert AmpliMASTER 

reagents optimized for real time amplification 

Complete size product, code RTS000 

MATERIALS PROVIDED IN THE PRODUCT 

RTS000 

RTS000/2 

«REAL TIME AMPLIFICATION ACCESSORIES» 



RTS000 

RTS000/2 

web site: www.nanogen.com 

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 Nanogen Advanced Diagnostics S.r.L. 

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

amplification method. 

PRODUCT DESCRIPTION 

+2° C 

+8° C 

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. 

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. 

SCH mRTS000_en 20/04/07 Review 01 Page 1/4 

Component Description Quantity Composition Labelling 

AmpliMASTER optimized reagent mixture 4 x 340 µl 

Microplate for amplification 

microplate 

with 96 x 0.2 ml wells 

TRIS base, TRIS hydrochloride, 

Glycerol, magnesium chloride, 

Deoxyribonucleotide triphosphates, 

ROX, Uracil-N-glycosidase, Taq 

DNA polymerase 

3 - - 

Adhesive sheet for amplification adhesive sealing sheet 3 - - 

Reduced size product, code RTS000/2 


AmpliMASTER optimized reagent mixture 2 x 340 µl 

Microplate for amplification 

microplate 

with 96 x 0.2 ml wells 


Glycerol, magnesium chloride, 

Deoxyribonucleotide triphosphates, 

ROX, Uracil-N-glycosidase, Taq 

DNA polymerase 

2 - - 

Adhesive sheet for amplification 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 knownquantity 

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 Nanogen Advanced 

Diagnostics S.r.L., 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 



RTS000 

RTS000/2 

This product is exclusively for in vitro use. 

Warnings and general precautions 

WARNINGS AND 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. 

Do not use reagents from other manufacturers' products. 

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. 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): 

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

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. 

Catalogue number. 

Temperature limits. 

Batch code. 

Use by (last day of month). 

In vitro diagnostic medical device. 

SYMBOLS 

In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic 

medical devices. 

Contents sufficient for "N" tests. 

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 Nanogen Advanced Diagnostics S.r.L. 

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. 


SCH mRTS000_en 20/04/07 Review 01 Page 4/4

«DUPLEX REAL TIME AMPLIFICATION» 

ENTEROVIRUS 

Q - PCR Alert AmpliMIX 

detection and dosing of Enterovirus cDNA 

RTS076-M 

Nanogen Advanced Diagnostics S.r.L. 



Offices: 

Tel. +39-011 976 19 1 

Fax +39-011 936 76 11 


web site: www.nanogen.com 



ASSAY PRINCIPLE page 2 






SAMPLES AND CONTROLS page 5 


PROCEDURE LIMITATIONS page 12 

PERFORMANCE CHARACTERISTICS page 13 

REFERENCES page 15 

TROUBLESHOOTING page 16 


INTENDED USE 

-20°C 

«ENTEROVIRUS Q - PCR Alert AmpliMIX» is part of a quantitative amplification assay of nucleic 

acids for the detection and dosing of Enterovirus cDNA in the product of reverse transcription reaction 

obtained from RNA extract in plasma samples collected in EDTA. 

The assay can detect and dose human Enterovirus cDNA belonging to serotypes: Poliovirus 1 - 3, 

Coxsackievirus A1 - A22 and A24, Coxsackievirus B1 - B6, Echovirus 1 - 9, 11 - 21, 24 - 27 and 29 - 33, 

Enterovirus 68 - 71. The assay cannot detect and dose human Parechovirus cDNA, previously known as 

Echovirus 22 and 23. 

The product is intended for use, alongside clinical data and other laboratory tests, in the diagnosis 

and monitoring of Enterovirus infections. 

ENTEROVIRUS Q - PCR Alert AmpliMIX 


ASSAY PRINCIPLE 

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The procedure involves a real-time amplification reaction on a microplate with programmable heater 

with optical fluorescence detection system (thermal cycler for real time). 

System standardization was carried out on Applied Biosystems ABI PRISM TM 7000 series 

instruments. 

In each well, an amplification reaction is carried out specific to the 5' UTR of the Enteroviruses and 

for a region of MS2 phage RNA genome (internal suitability test of the sample) using the cDNA produced in 

the retrotranscription reaction of the samples being tested. A specific probe for Enterovirus labelled with FAM 

fluorophor is activated when hybridized with the specific product of the Enterovirus amplification reaction. 

Another probe specific for MS2 phage labelled with VIC fluorophor is activated when hybridized with the 

product of the MS2 phage amplification reaction. Fluorescence emission increases as the specific product of 

the amplification reaction increases and is measured and recorded by the apparatus. 

The processing of the data determines the presence and the titre of Enterovirus cDNA in the starting 

sample. 


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 tubes. Each test tube contains 110 µl 

of solution, sufficient for 24 tests. 

The primer oligonucleotides for Enterovirus are specific for the 5' UTR region of Enterovirus. 

The primer oligonucleotides for the internal suitability test of the sample are specific for a region of 

MS2 phage RNA genome . 

The product provides 96 determinations, including standards and controls. 



ENT. AmpliMIX 

primer oligonucleotides 

mixture 

4 x 110 µl 

Oligonucleotides, 


Glycerol, Triton X-100 



- Disposable latex gloves or similar material. 

- Vortex mixer. 



50-200 µl, 200-1000 µl). 



- 

SCH mRTS076M_en 15/05/06 Review 00 Page 1/17 

SCH mRTS076M_en 15/05/06 Review 00 Page 2/17



RTS076-M 



RTS076-M 


The reagents for RNA extraction from the samples to be analysed, the positive extraction control, the 

reagents for reverse transcription, the reagents optimized for amplification, the detection reagents 

(fluorescent probes) and the positive amplification control or the known-quantity DNA standard are not 

included in this product. To perform these analytical steps the following accessory products are 

recommended, manufactured by Nanogen Advanced Diagnostics S.r.L.: 

«EXTRAgen ® » (code EXTG01), kit for extraction of nucleic acids from non-cellular samples; the kit 

enables 50 extractions. 

«CPE - RNA ® - Internal Control» (code CTRRNA), positive RNA extraction control for non-cellular 

sample RNA extraction systems; the kit enables 50 extractions. 

«RT - kit plus» (code BRK200), kit for reverse transcription of RNA with random primer; the kit 

enables 50 reactions. 

«Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates 

and adhesive sheets for real-time amplification; the product provides 96 reactions. 

«ENTEROVIRUS Q - PCR Alert AmpliPROBE» (code RTS076-P), fluorescent probes for real-time 

amplification; the product provides 96 reactions. 

If a qualitative result of the analysis is required (detection of Enterovirus cDNA): 

«ENTEROVIRUS - Positive Control» (code CTR076), positive amplification control of plasmid DNA; 

the product enables 25 sessions. 

If a quantitative result of the analysis is required (dosing of Enterovirus cDNA): 

«ENTEROVIRUS Q - PCR Standard» (code STD076), known-quantity plasmid DNA to obtain the 

standard curve; the product enables 16 sessions. 








Handle and dispose of all reagents and all assay materials as if they were potentially infective. 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. 






































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. 




RTS076-M 



RTS076-M 

SAMPLES AND CONTROLS 

PROCEDURE 

Samples 

The material used with this product must consist of the product of the reverse transcription reaction 

(cDNA) obtained from the RNA extracted from biological samples. 

The RNA reverse transcription system must use random primers to trigger the polymerization 

reaction. 

The system for RNA extraction from the starting sample must provide RNA that is suitable for 

reverse transcription and amplification reactions. 

The biological samples to be used for RNA extraction must be collected and stored as described 

below: 

- plasma collected in EDTA, must be stored at +2° / +8°C for a maximum of four hours or frozen at 

-20°C for a maximum of thirty days or at -70°C for longer periods. 

Split the samples that are to be stored frozen into aliquots in order to prevent repeated cycles of 

freezing and thawing. 

Instructions for pre-treatment of clinical samples, where applicable, and for DNA extraction are 

contained in the manual for «EXTRAgen ® ». 

Interfering substances 

The cDNA produced by the reverse transcription of the RNA extracted from the starting sample must 

not contain heparin or haemoglobin in order to prevent problem of inhibition and the possibility of frequent 

invalid results. 

There are no data available concerning inhibition caused by antiviral 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 cDNA obtained from the sample. 

For the positive control, use «ENTEROVIRUS - Positive Control» product or «ENTEROVIRUS 

Q - PCR Standard» product. 

Quality controls 

It is recommended to validate the whole analysis procedure of each extraction and amplification 

session by processing a negative sample and a positive sample that have already been tested or calibrated 

reference material. 

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 software and open an "absolute quantitation" session; 

- referring to the instrument documentation, set the "detector" for the Enterovirus probe with the 

"reporter" as "FAM" and the "quencher" as "none" (NFQ = non-fluorescent quencher); 

- referring to the instrument documentation, set the "detector" for the beta globin 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, knownquantity 

standard). 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. 

N.B.: In order to determine the DNA titre in the starting sample, set up a series of reactions with the Q - PCR 

standards (10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies) to obtain the standard curve. 

Below is an example of how the analysis of 11 samples can be organized. 

C1 C9 

C2 C10 

C3 C11 

C4 CN 

C5 10 2 

C6 10 3 

C7 10 4 

C8 10 5 

Key: C1 - C11: Samples to be analysed; CN: Negative amplification control; 

10 2 : Standard 10 2 copies; 10 3 : Standard 10 3 copies; 10 4 : Standard 10 4 copies; 10 5 : Standard 10 5 copies. 

- set the parameters of the heat cycle on the thermal cycler and a reaction volume of 25 µl. For 

Applied Biosystems ABI PRISM TM instruments of the 7000 series choose the "9600 emulation" 

option. 

Amplification thermal cycle 

Phase Temperature Timing 

Decontamination 50° C 2 min. 

Initial denaturation 95° C 10 min. 

45 cycles 

95° C 15 sec. 

60° C 1 min. 





RTS076-M 



RTS076-M 

Amplification set-up 

(To be performed in the amplification /detection area of the amplification products) 

Before starting the extraction 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 

"ENT." 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 tube or the Q - PCR Standard tubes. Centrifuge the 

tubes 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. Gently deposit 20 µl of the reaction mixture obtained in this way on the bottom of the 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 "ENT." Freeze and thaw the reaction mixture ONLY ONCE. 

6. Gently deposit 5 µl of DNA extract 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. Gently deposit 5 µl of sterile bidistilled water (not supplied with the product) on the bottom of the 

negative control microplate well, as previously established on the Work Sheet. 

8. Gently deposit 5 µl of Positive Control in the reaction mixture in the corresponding amplification 

microplate well, as previously established on the Work Sheet. 

N.B.: When this product is used for dosing Enterovirus cDNA, carefully deposit 5 µl of Q - PCR Standard 

10 2 copies in the reaction mixture in the corresponding amplification microplate well as previously 

established on the Work Sheet. Proceed in this way for the other Q - PCR Standard (10 3 , 10 4 , 10 5 copies). 

9. Carefully seal the amplification microplate using the amplification adhesive sheet. 

10. Transfer the microplate to the real-time thermal cycler in the amplification/detection area of the 

amplification products and start the thermal amplification cycle. 

Qualitative analysis of the results 

The values of fluorescence emitted by the specific probe for Enterovirus (FAM fluorescence) and by 

the specific probe for the MS2 phage (VIC fluorescence) in the amplification reactions must be analysed by 

the instrument software. 

Before analysing, it is necessary to: 

- referring to the instrument documentation, manually set the calculation range for the fluorescence 

back ground level (Baseline) from cycle 6 to cycle 15*; 

*N.B.: in the case of a positive sample with a high titre of Enterovirus the FAM fluorescence of the specific 

probe for the Enterovirus may begin to grow before the 15th cycle. In this case the calculation range for the 

“baseline” must be adjusted from cycle 6 to the cycle in which the FAM fluorescence starts to grow. 

- Referring to the instrument documentation, manually set the Threshold for the FAM fluorescence 

to 0.2; 

- Referring to the instrument documentation, manually set the Threshold for the VIC fluorescence to 

0.1. 

The values of fluorescence emitted by the specific probe for Enterovirus in the Positive control 

amplification reaction and the Threshold value of fluorescence are used to validate amplification and 

detection as shown in the following table: 

Enterovirus Positive control 

Threshold cycle (FAM) 

Assay result 

Amplification / Detection 

Determined POSITIVE CORRECT 

If the result of the Positive control amplification reaction is Undetermined, it means that target 

DNA has not been detected. A problem has occurred during the amplification or detection phase (incorrect 

reaction mixture volumes, probe degradation, positive control degradation, incorrect dispensing of positive 

control, incorrect positive control position setting, incorrect thermal cycle setting) which may cause incorrect 

results. The session is invalid and must be repeated from the amplification phase. 

N.B.: When this product is used for dosing Enterovirus cDNA, amplification and detection must be validated 

in relation to the value of fluorescence emitted by the specific probe for Enterovirus in the amplification 

reactions of the four Q - PCR Standards, used instead of the Positive Control, and to the fluorescence 

Threshold value. 

The values of fluorescence emitted by the specific probe for Enterovirus in the negative control 

amplification reactions and the threshold value of fluorescence established for the amplification session are 

used to validate amplification and detection as shown in the following table: 

Negative control threshold cycle 

Enterovirus (FAM) 

Assay result 

Amplification 

Undetermined NEGATIVE CORRECT 

If the result of the negative control amplification reaction is anything other than undetermined, i.e. 

if target DNA has been detected in the amplification reaction through determination of the cycle threshold 

(Ct), in which the threshold value of fluorescence has been reached, it means that a problem has occurred 

during the amplification phase (contamination) which may cause incorrect results and false positives. 

The session is invalid and must be repeated from the amplification phase. 





RTS076-M 



RTS076-M 

The values of fluorescence emitted by the probes in the amplification reactions of each sample and 

the threshold value of fluorescence are used to detect the presence of target cDNA and to validate 

amplification and detection by determining the threshold cycle. 

This product is able to detect a minimum quantity of cDNA molecules in the 5' UTR of Enteroviruses 

by amplification reaction, equivalent, per reaction, to at least 10 genome copies (see paragraph on 

Performance Characteristics on page 13). 

When this product is used for detection of Enterovirus, the results for each sample are used as 

shown in the following table: 

Threshold cycle of the sample 

Enterovirus (FAM) 

Undetermined 

Determined 

MS2 (VIC) 

Ct > 35 or 

Undetermined 

Sample 

suitability 

Assay result 

Enterovirus 

cDNA 

not suitable invalid - 

Ct ≤ 35 suitable valid, negative NOT DETECTED 

Ct > 35 or 

Undetermined 

suitable* valid, positive PRESENT 

Ct ≤ 35 suitable valid, positive PRESENT 

If the result of the negative control amplification reaction is undetermined for the cDNA of the 5' 

UTR of Enterovirus and Ct > of 35 or undetermined for the cDNA of the MS2 phage, i.e. if an insufficient 

quantity of target cDNA has been detected in the amplification reaction or in the cDNA of MS2 phage, it 

means that a problem has occurred during the amplification phase (inefficient or invalid amplification), in the 

reverse transcription phase (inefficient or invalid reverse transcription) or in the extraction phase (loss of 

RNA, presence of inhibitors, degradation of the RNA sample or insufficient number of cells in the starting 

sample) which may cause incorrect results and false positives. 

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 undetermined for the cDNA of the 5' UTR of 

Enterovirus and determined for the cDNA of MS2 phage, the Enterovirus cDNA has not been detected in 

the product of the reverse transcription reaction obtained from the RNA extracted from the sample. 

The sample can be considered negative, but it may also contain Enterovirus cDNA at a lower titre 

than the detection limit for the product (see paragraph on Performance Characteristics on page 13). In this 

case the result would be a false negative. 

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. 

*N.B.: When Enterovirus cDNA has been detected in the amplification reaction of a sample, the amplification 

of the MS2 phage cDNA may result in Ct > 35 or undetermined. In fact, the low-efficiency amplification 

reaction of the MS2 phage cDNA may be cancelled out by competition with the high-efficiency amplification 

reaction of the Enterovirus cDNA. In this case the sample is still suitable and the positive result of the assay 

is valid. 

Quantitative analysis of the results 

After carrying out the procedure for qualitative analysis of the results it is possible to perform the 

quantitative analysis of the results of the positive samples. 

The values of fluorescence emitted by the specific probe for Enterovirus in the amplification reactions 

of the four Q - PCR Standard are used to calculate the Standard Curve of the amplification session and to 

validate the amplification and detection as shown in the following table: 

Enterovirus Standard Curve 

(FAM) 

Acceptance range 

Amplification / Detection 

Correlation coefficient (R2) 0.990 ≤ R2 ≤ 1.000 CORRECT 

If the Correlation coefficient value (R2) is not within the limits, it means that a problem has 

occurred during the amplification or detection phase (incorrect reaction mixture volumes, probe degradation, 

standard degradation, incorrect dispensing of the standards, incorrect standard position setting, incorrect 

thermal cycle setting) which may cause incorrect results. The session is invalid and must be repeated from 

the amplification phase. 

The values of fluorescence emitted by the specific probe for Enterovirus in the amplification reactions 

of each sample and the standard curve of the amplification session are used to calculate the quantity of 

target cDNA present in the amplification reactions of the samples. 

This product is able to measure a quantity of cDNA molecules in the 5' UTR of Enteroviruses by 

amplification reaction, equivalent, per reaction, to between 1,000,000 and 10 genome copies (see paragraph 

on Performance Characteristics on page 13) as shown in the following table: 

Result of the Enterovirus 

sample (FAM) 

Equivalent Enterovirus genomes per reaction 

Quantity > 1 x 10 6 GREATER THAN 1,000,000 

1 x 10 1 ≤ Quantity ≤ 1 x 10 6 = Quantity 

Quantity < 1 x 10 1 FEWER THAN 10 

When this product is used for quantitative measuring of Enterovirus, the results (Quantity) of the 

amplification reactions on the samples are used to calculate the number of genome equivalents (gEq) of 

Enterovirus available in the starting sample (Nc) according to this formula: 

Ve x Vp X Quantity 

Nc (gEq / ml) = ————————————— 

Vc x Vt x Va x Ee Et 

Vc is the volume of the sample used in the extraction; for example with «EXTRAgen ® » the Vc 

parameter is 0.3 ml. 

Ee is the efficiency of the extraction; for example with «EXTRAgen ® » the Ee parameter is 0.8 

(minimum efficiency of 80%). 

Ve is the total volume of the extraction product; for example with «EXTRAgen ® » the Ve parameter is 

15 µl. 

Vt is the volume of reverse transcription product, for example with «RT - kit plus» product, the Vt 

parameter is 10 µl. 

Et is the efficiency of reverse transcription reaction, for example with «RT - kit plus» product, the Et 

parameter is 0,5 (minimum efficiency of 50%). 

Vp is the total volume of reverse transcription reaction, for example with «RT - kit plus» product, the 

Vp parameter is 25 µl 

Va is the volume of the extraction product used in the reverse transcription reaction: with this product 

the Va parameter is 5 µl. 

Quantity is the result of the amplification reaction of the sample in gEq. 





RTS076-M 



RTS076-M 

When Nanogen Advanced Diagnostics S.r.L. extraction kits are used, the formula becomes: 

Extraction kit 

«EXTRAgen ® » 

«RT - kit plus» 

Calculation of the dosing range limits 

Simplified formula 

Nc (gEq / ml) = 62.5 x Quantity / ml 

When a particular extraction assay method is used, the dosing range limits may be calculated from 

the dosing range of the amplification reaction according to the following formula. 

Ve x Vp x 10 gEq 

Lower limit (gEq / ml) = ————————————— 

Vc x Vt x Va x Ee x Et 

Ve x Vp x 1.000.000 gEq 

Upper limit (gEq/ml) = —————————————— 

Vc x Vt x Va x Ee x Et 

When Nanogen Advanced Diagnostics S.r.L. extraction and reverse transcription reaction kits are 

used, the formula becomes 

Products 

«EXTRAgen ® » 

«RT - kit plus» 

Dosing range limits 

from 625 to 62.500.000 gEq / ml 

PROCEDURE LIMITATIONS 

With this product, use only the cDNA produced by the reverse transcription of the RNA extracted 

from the following human samples: plasma collected in EDTA 

With this product, do not use the cDNA produced by the reverse transcription of the RNA extracted 

from heparinized samples: heparin inhibits the reverse transcription and amplification reactions of nucleic 

acids and causes invalid results. 

With this product, do not use the cDNA produced by the reverse transcription of haemoglobincontaminated 

RNA: haemoglobin inhibits the reverse transcription and amplification reactions of nucleic 

acids and may causes invalid results. 

There are no data available concerning inhibition caused by antiviral 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 Enterovirus-positive clinical samples, positive controls and the 

amplification reaction products themselves. Contamination leads to false positive 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 molecular biology techniques, such as 

extraction, reverse transcription, amplification and detection of nucleic acids, to avoid incorrect results. 


the amplification/detection of amplification products to prevent false positive 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 false positive results. 

A false negative result obtained with this product suggests that the Enterovirus cDNA was not 

detected in the reverse transcription product obtained from the RNA extracted from the sample, but it may 

also contain Enterovirus cDNA at a lower titre than the detection limit for the product (see paragraph on 

Performance Characteristics on page 13); in this case the result would be a false negative. 

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 results, false positives and 

false negatives with this product. This risk cannot be eliminated or reduced any further. In particular 

situations such as emergency diagnoses, this residual risk can contribute to incorrect decisions with 

potentially grave consequences for the patient. 





RTS076-M 



RTS076-M 

Analytical sensitivity: Detection limit 

PERFORMANCE CHARACTERISTICS 

The analytical sensitivity of this assay enables identification of c. 10 target cDNA molecules in 5 µl of 

product obtained by reverse transcription of RNA extract added to the amplification reaction. 

In terms of the detection limit, the analytical sensitivity of the assay was tested using plasmid DNA 

containing the amplification product whose initial concentration was measured by spectrophotometer. The 

plasmid DNA was diluted to a titre of 10 copies / 5 µl. This sample was used in 50 repeats for amplification 

with Nanogen Advanced Diagnostics S.r.L. products (see paragraph on accessory products). 

The final results are summed up in the following table. 

Samples N negative Positive 

10 copies plasmid DNA + MS2 phage cDNA 50 0 50 

Analytical sensitivity: Linear measuring range 

In terms of the linear measuring range, the analytical sensitivity of this assay enables detection of a 

titre of between 1,000,000 and 10 target DNA molecules in 5 µl of cDNA produced in the reverse 

transcription reaction added to the amplification reaction. 

In terms of the linear measuring range, the analytical sensitivity of the assay was determined using a 

panel of dilutions (1 log10 between one dilution and the next) of plasmid DNA containing the amplification 

product whose initial concentration was measured by spectrophotometer. The panel points from 10 7 

molecules per reaction to 10 1 molecules per reaction were used in 9 repeats for amplification with Nanogen 

Advanced Diagnostics S.r.L. products. The analysis of the data obtained, performed via linear regression, 

demonstrated that the assay has a linear response for all the panel points (linear correlation coefficient 

greater than 0.99). 


Linear measuring range 

cDNA copies/ reaction 

gEq / ml 

Upper limit 1,000,000 62,500,000 

Lower limit 10 625 

The upper limit of the linear measuring range was fixed at 10 6 molecules / 5 µl, within one logarithm 

of the highest concentration Q - PCR Standard amplification standard (10 5 molecules / 5 µl). 

The lower limit of the linear measuring range was fixed at 10 molecules / 5 µl, within one logarithm of 

the lowest concentration Q - PCR Standard amplification standard (10 2 molecules / 5 µl). 

Diagnostic sensitivity: efficiency of detection on different genotypes / subtypes 

The diagnostic sensitivity of the assay, that is the efficiency of detection and quantitation on different 

genotypes / subtypes, was evaluated by comparison of sequences with nucleotide databases. 

The alignment test of the regions chosen for hybridization of the AmpliMIX primer oligonucleotides 

and of the AmpliPROBE fluorescent probe with the sequences available in the database of the 5' UTR of 

human Enterovirus showed preservation and absence of significant mutations. 

The diagnostic sensitivity of the assay, that is the efficiency of detection on different genotypes / 

subtypes, was tested using positive samples for Coxsackievirus A9, Coxsackievirus B5 and Echovirus 11. 

The results are shown in the table in the paragraph entitled "Tests with reference materials: panel 

QCMD 2003 Enterovirus Proficiency Programme" on 14. 

Diagnostic specificity: negative samples 

The diagnostic specificity of the assay, confirming negative clinical samples, was tested by analysing 

a panel of normal donor plasma samples and proved to be greater than 92%. 

Diagnostic specificity was evaluated using a panel of 12 normal donor plasma samples (Panel 

Normal Human Plasma, VQC, the Netherlands). Each panel sample was used in two repeats to carry out the 

entire procedure for analysis, extraction, and amplification with Nanogen Advanced Diagnostics S.r.L. 

products. 


Samples N Negative Positive 

Panel of normal donor plasma 12 12 0 

Analytical specificity: potentially interference markers 

The analytical specificity of the assay, that is the cross-reactivity with other potential interference 

markers, was evaluated by comparison of sequences with nucleotide databases. 

The alignment test of the regions chosen for hybridization of the AmpliMIX primer oligonucleotides 

and of the AmpliPROBE fluorescent probe with the sequences available in databases of human organisms 

other than human Enteroviruses, including Parechoviruses and Rhinoviruses, the human viruses that are 

most similar to Enteroviruses, showed the occurrence of significant homology only in a few serotypes of 

human Rhinovirus, and particularly Rhinovirus 87. 

The analytical specificity of the assay, that is the cross-reactivity with other potential interference 

markers, was tested using a positive sample for Parechovirus 1d and a positive sample for Rhinovirus type 

16, the human viruses most similar to Enterovirus. The results are shown in the table in the paragraph 

entitled "Tests with reference materials: panel QCMD 2003 Enterovirus Proficiency Programme" on 14. 

Analytical sensitivity: Precision 

The assay precision study, that is, the variability of the results in different repeats of a sample with 

the same concentration analysed during the same session, made it possible to determine a coefficient of 

variation (CV %) of 12.5% within the linear range of 10 6 molecules / 5 µl to 10 molecules / 5 µl. 

Analytical sensitivity: Accuracy 

The assay accuracy study, that is, the difference between the mean results obtained in a single 

session on different repeats of a sample with the same concentration and the theoretical value of the 

concentration of the samples, made it possible to determine a mean percentage inaccuracy of 9.0% within 

the linear range of 10 6 molecules / 5 µl to 10 molecules / 5 µl. 





RTS076-M 



RTS076-M 

Tests with reference materials: panel 2003 QCMD Enterovirus Proficiency Programme 

The diagnostic sensitivity and the analytical specificity of the assay were tested using an Enterovirus 

panel as the reference material (QCMD 2003 Enterovirus Proficiency Programme). 

The panel samples were used in two repeats to carry out the entire procedure for analysis, 

extraction, and amplification with Nanogen Advanced Diagnostics S.r.L. products. 


Test 1 Test 2 Expected 

Sample Serotype TCID50/ml Dilution 

gEq/ml gEq/ml result 

EV03-01 RHINO 16 3.2 1:10 3 Not found Not found - 

EV03-02 N/A (Negative) N/A N/A Not found Not found - 

EV03-03 N/A (Negative) N/A N/A Not found Not found - 

EV03-04 COX A9 0.03 1:10 8 < 625 (108) Not found +/- 

EV03-05 COX A9 0.3 1:10 7 936 1,659 + 

EV03-06 ECHO 11 250 1:10 5 < 625 (231) < 625 (62) + 

EV03-07 COX A9 30 1:10 5 90,044 89,215 + 

EV03-08 COX B5 32 1:10 6 < 625 (289) < 625 (208) + 

EV03-09 PARECHO 1d 32000 1:10 2 Not found Not found - 

EV03-10 COX A9 3 1:10 6 5,657 5,569 + 

EV03-11 ECHO 11 25 1:10 6 invalid* invalid* + 

EV03-12 COX B5 320 1:10 5 1,025 1,003 + 

*Sample EV03-011, a 1:10 6 dilution of Echovirus 11, inhibited amplification in both tests. 

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 "ENTEROVIRUS Q - PCR Alert 

AmpliMIX" and "ENTEROVIRUS Q - PCR Alert AmpliPROBE", FTP RTS076. 

REFERENCES 

TROUBLESHOOTING 

Target DNA not detected in the Positive Control / Q - PCR Standard reaction or 

invalid correlation coefficient of the standard curve. 

Possible causes 

Solutions 

Check the volumes of reagent dispensed during 

Error in the preparation of the reaction mixture. 


Take care when dispensing reactions onto the microplate 

and comply with the work sheet. 

Dispensing error on the microplate. 

Check the volumes of reaction mixture dispensed. 

Check the volumes of standard dispensed. 

Probe degradation. 

Positive control or standard degradation. 

Instrument setting error. 

Target DNA detected in the negative control reaction 


Dispensing error on the microplate. 

Error while setting the instrument 

Microplate badly sealed. 

Use a new probe aliquot. 

Use a new aliquot of Positive control or standard. 

Check the position settings for the positive control or 

standard reactions on the instrument. 

Check the thermal cycle settings on the instrument. 

Solutions 

Avoid spilling the contents of the sample test tube. 

Always change tips between one sample and another. 

Take care when dispensing samples, negative controls, 

positive controls and standards onto the microplate and 

comply with the work sheet. 

Check the position settings of the samples, negative 

controls, positive controls and standards on the instrument 

Take care when sealing the microplate. 

W. A. Verstrepen et al. (2001) J Clin Microbiology 39: 4093 – 4096 

Contamination of the sterile bidistilled water. 

Use a new aliquot of sterile water. 

Contamination of the amplification mix. 

Contamination of the extraction/preparation 

area for amplification reactions. 

High levels of background fluorescence in the reactions 

Use a new aliquot of amplification mix. 

Clean surfaces and instruments with aqueous detergents, 

wash lab coats, replace test tubes and tips in use. 

Baseline setting error. 


Solutions 

Set baseline calculation interval within cycles where the 

background fluorescence has already stabilized (check the 

"component" recordings) and where the signal 

fluorescence has not started to grow yet, e.g. from cycle 9 

to cycle 15. 






SYMBOLS 

RTS076-M 

Nanogen Advanced D iagnostics S.r.L. 



Offices: 

Tel. +39-011 976 19 1 

Fax +39-011 936 76 11 


website: www.nanogen.com 

Upper temperature limit. 

Batch code. 


«DUPLEX REAL TIME AMPLIFICATION» 

ENTEROVIRUS 

Q - PCR Alert AmpliPROBE 


In vitro diagnostic medical device: 



RTS076-P 

-20°C 



Manufacturer. 









REFERENCES page 4 


INTENDED USE 

«ENTEROVIRUS Q - PCR Alert AmpliPROBE» is part of a quantitative amplification assay of 

nucleic acids for the detection and dosing of Enterovirus cDNA in the product of reverse transcription 

reaction obtained from RNA extract in plasma samples collected in EDTA. 

The product is intended for use, alongside clinical data and other laboratory tests, in the diagnosis 

and monitoring of Enterovirus infections. 




licensed products for "Positive Control" or "Q - PCR Standard". 



The product supplies the mixture of AmpliPROBE fluorescent probes for real-time amplification in a 

stabilizing solution, pre-dosed in aliquots into disposable test tubes. Each tubes contains 110 µl of 

solution, sufficient for 24 tests. 

The Enterovirus probe, labelled with FAM fluorophor and blocked by the MGB-NFQ group, is specific 

for the 5' UTR of Enterovirus. 

The MS2 phage probe, labelled with VIC fluorophor and blocked by the MGB-NFQ group, is specific 

for a region of MS2 phage RNA genome. 

The procedure involves a real-time amplification reaction on a microplate with programmable heater 

with optical fluorescence detection system (thermal cycler for real time). 

System standardization was carried out on Applied Biosystems ABI PRISM TM 7000 series 

instruments. 

SCH mRTS076P_en 15/05/06 Review 00 Page. 1/4

ENTEROVIRUS Q - PCR Alert AmpliPROBE 


RTS076-P 



RTS076-P 

The product provides 96 determinations, including standards and controls. 



ENT. AmpliPROBE 

mixture of fluorescent probes 

labelled with FAM / MGB-NFQ 

and VIC / MGB-NFQ 

4 x 110 µl 

fluorescent oligonucleotides, 


Glycerol, Triton X-100 



- Disposable latex gloves or similar material. 

- Vortex mixer. 



50-200 µl, 200-1000 µl). 




The reagents for RNA extraction from the samples to be analysed, the positive extraction control, the 

reagents for reverse transcription, the reagents optimized for amplification, the primer reagents 

(oligonucleotides), the positive amplification control or the known-quantity DNA standards are not included in 

this product. To perform these analytical steps the following accessory products are recommended, 

manufactured by Nanogen Advanced Diagnostics S.r.L.: 

«EXTRAgen ® » (code EXTG01), kit for extraction of nucleic acids from non-cellular samples; the kit 


«CPE - RNA ® - Internal Control » (code CTRRNA), positive RNA extraction control for non-cellular 

sample RNA extraction systems; the kit enables 50 extractions. 

«RT - kit plus» (code BRK200), kit for reverse transcription of RNA with “random primer”; the kit 


«Q - PCR Alert AmpliMASTER» (code RTS000), combination of optimized reagents, microplates 

and adhesive sheets for real-time amplification; the product provides 96 reactions. 

«ENTEROVIRUS Q - PCR Alert AmpliMIX» (code RTS076-M), primer oligonucleotides for real-time 

amplification; the product provides 96 reactions. 

If a qualitative result of the analysis is required (detection of Enterovirus cDNA): 

«ENTEROVIRUS - Positive Control» (code CTR076), positive amplification control of plasmid DNA; 

the product enables 25 sessions. 

If a quantitative result of the analysis is required (dosing of Enterovirus cDNA): 

«ENTEROVIRUS Q - PCR Standard» (code STD076), known-quantity plasmid DNA to obtain the 

standard curve; the product enables 16 sessions. 








SCH mRTS076P_en 15/05/06 Review 00 Page 2/4 

- 

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. 





































The test tubes containing AmpliPROBE are disposable and therefore must be used once only in the 


The AmpliPROBE carries the following safety warnings (S): 

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

SCH mRTS076P_en 15/05/06 Review 00 Page 3/4



RTS076-P 

PROCEDURE 

The «ENTEROVIRUS Q - PCR Alert AmpliPROBE» product must be used with «Q - PCR Alert 

AmpliMASTER» and «ENTEROVIRUS 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 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 instructions manual enclosed with the «ENTEROVIRUS Q - PCR Alert 

AmpliMIX» kit. 

The performance characteristics and procedure limitations of the complete assay for detection and 

dosing of Enterovirus cDNA are described in detail in the instructions manual enclosed with the 

«ENTEROVIRUS Q - PCR Alert AmpliMIX» kit. 

REFERENCES 

W. A. Verstrepen et al. (2001) J Clin Microbiology 39: 4093 – 4096 


SYMBOLS 

Upper temperature limit. 

Batch code. 


In vitro diagnostic medical device: 





Manufacturer. 



licensed products for "Positive Control" or "Q - PCR Standard". 


SCH mRTS076P_en 15/05/06 Review 00 Page 4/4

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