Reference: 67-080

Intended use

• The ENTEROVIRUS CONSENSUS kit allows the specific genome detection in cerebrospinal fluid (CSF), throat specimens, nasopharyngeal secretions and stool specimens using hybridization in a microtiter plate with a biotinylated probe after amplification of Enterovirus genome. This kit is for research use only.

COMPOSITION: Kit for purification of viral RNA, QIAamp® Viral RNA Mini Kit

Ref.: 67-080A

Amplification kit

Ref.: 67-080B

Detection kit

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Ref.: 67-080C

Intended use. Presentation of the kit. Principle of the test. Reagents and materials supplied. Material and reagents required but not supplied. Reagents reconstitution. Warning and precautions. Sample treatment and transport. Controls. Sample extraction protocol. Reverse transcription and amplification protocol. Detection protocol. Calculation and results interpretation. Trouble shooting guide. Performances of the test. References. Outlined procedure.

2.

Presentation of the kit

• This kit allows the detection of the conserved part of the Enterovirus genome. Enteroviruses, members of the Picornaviridae family are single stranded RNA viruses. Sixty four serotypes are identified including Poliovirus 1-3, Coxsackievirus A1-22, A24, Coxsackievirus B1-6, Echovirus 1-9, 11-21, 24-27, 29-33 and Enterovirus 68-71. Recently reclassified in the Parechovirus genus, Echovirus 22 and 23 are not detectable with this kit. • In temperate climates Enterovirus infections occur seasonally and are particularly common infections in children and adolescents. Enteroviruses are often associated as causal agents of meningitis. The clinical symptoms are somewhat non-specific, which makes infection difficult to diagnose because of their similarity to infections with other causal agents. Enterovirus infections have been also associated with cardiopathic, respiratory disorders, mucouscutaneous pathologies and febrile disease in neonates. The Poliovirus group is most closely associated with Poliomyelitis. • Diagnosis of Enteroviruses is often done by viral culture isolation with typing by sero-neutralization. This traditional approach does not allow the detection of all serotypes, particularly Coxsackievirus group A, which does not grow in culture. Moreover grow of Enterovirus from CSF samples is not always successfuly incubated in cell culture. In addition, cell culture techniques are time consuming, require several days incubation. • The Enterovirus genome detection by consensus methodologies allows rapid and sensitive results.

3.

Principle of the test

3.1 Viral RNA extraction from samples • Viral RNA is extracted with the QIAamp® Viral RNA Blood Mini Kit which associates the selective binding properties of silica gels with a microcentrifugation step. • The sample is first lysed in highly denatured conditions in order to inactivate the RNases and obtain free viral RNA. A specific buffer is then added to the RNA in order to optimize the membrane binding capacities. Once the RNA is fixed, use of a silica column allows washing of the sample to eliminate contaminants. The elution is performed with an RNases-free buffer.

3.2 Amplification • The reverse transcription and amplification steps are performed in two parts. The chosen primers allow the amplification of a sequence with 3’ mutation with good efficiency. For the Enterovirus group, detection of all serotypes as listed is performed with a single amplification. The amplified RNA is located in the 5’ non-coding conserved portion of the genome. The size of the amplified fragment is 425 bp. • A positive control corresponding to a plasmid with Coxsackie B4 virus sequence for the primer sequence is used as an amplification positive sample. The absence of inhibitors is demonstrated through the use of this positive control (included in the Inhibition control premix) tested with a duplicate sample after RNA extraction.

3.3 Detection • After amplification, the amplified products are chemically denatured and analyzed by hybridization in a microtiter plate. The hybridization is performed with a biotinylated probe which is Enterovirus groupspecific. Detection is then performed with a streptavidine peroxidase conjugate associated with 3,3’,5,5’ tetramethylbenzidine TMB). The optical density (OD) is read at 450 nm with a microplate reader.

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ＣＯＳＭＯ ＢＩＯ ＣＯ.,ＬＴＤ.

FOR RESEARCH USE ONLY

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67-080 - 05/09/01 - 1/8

1.

ENTEROVIRUS CONSENSUS

1

Reagents and materials supplied

4.1 Kit for purification of viral RNA, Preparations per kit: 50 QIAamp® mini column Collection tubes (2 mL) AVL buffer AW1 buffer (concentrate) AW2 buffer (concentrate) AVE buffer Carrier RNA

5 X 10 4 X 50 31 mL 19 mL 13 mL 3 X 2 mL 310 µg

• This kit can be stored dry at room temperature (+18°C/+25°C) until the expiry date written on the box. Storage at higher temperatures should be avoided.

67-080B

4.2 Amplification kit Reverse transcription assays: 50 Amplification assays: 100 Reverse transcription premix

2 X 200 µL

Contains primers, buffer, dNTPs used for the reverse transcription step

R9 R10 R11

Material and reagents required but not supplied

5.1 Kit for purification of viral RNA,

67-080A

QIAamp® Viral RNA Mini Kit

R8

5.

Reverse transcriptase Omniscript™ (4U/µL)* RNase inhibitor (40U/µL) Amplification premix----------------------------------------

200 U X 50 µL 800 U / 20 µL 3 X 650 µL

QIAamp® Viral RNA Mini Kit

67-080B

• Micropipettes with plugged (aerosol barrier) tips or positive displacement tips. • Thermal cycler. • Single use latex or similar gloves. • Sterile water. • 0.2 mL polypropylene tubes for amplification. • HotStarTaq™ QIAGEN, ref.: 203203 (250 U) at 5 Units/µL.

Contains primers, buffer, MgCl2, dNTPs

R12

Inhibition control premix-----------------------------------

Contains primers, buffer, MgCl2, dNTPs, inhibition control * manufactured by QIAGEN

Xn - HARMFUL

3 X 650 µL

• Keep the kit frozen at -18°C/-22°C until expiry date. The reagent R12 must be stored in the extraction room at -18°C/-22°C until expiry date.

67-080C

4.3 Detection kit

Number of detections with the two probes: 48 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26

Detection negative control Denaturation solution 1 Denaturation solution 2 Coating solution Microtiter plate (12 x 8 wells) Enterovirus generic probe ready to use (red) --------Control probe ready to use (blue)-----------------------Washing solution (10x) Conjugate diluent Streptavidine peroxidase conjugate (50x) Substrate - Tetramethylbenzidine (TMB) Stop solution Self adhesive cover foils

T - TOXIC

120 µL 1.5 mL 1.5 mL 35 mL 2 8 mL 8 mL 2 x 60 mL 15 mL 0.3 mL 15 mL 20 mL 4

5.3 Detection kit

Reagents reconstitution

6.1 Kit for purification of viral RNA,

67-080A

• Ethanol 96-100%. • Centrifuge. • Polypropylene test tubes (1.5 mL, 2 mL). • Incubator +80°C. • Micropipettes with plugged (aerosol barrier) tips or positive displacement tips, RNase-free. • Single use latex or similar gloves.

5.2 Amplification kit

6.

67-080C

• Microtiter plate reader (450 or 450/650 nm) and printer. • Automated microtiter plate washer (recommanded but not required). • Sterile pipettes. • Micropipettes with plugged (aerosol barrier) tips or positive displacement tips. • Multichannel pipettor. • Disposable reagent reservoirs. • Polypropylene tubes (1.5 mL and 15 mL). • Incubator (+37°C). • Vortex mixer. • Distilled water. • Single use latex or similar gloves.

67-080A

QIAamp® Viral RNA Mini Kit

6.1.1 Addition of carrier RNA to buffer AVL • Check buffer AVL for precipitate, and if necessary incubate at +80°C until the precipitate is dissolved. • Add 1 mL of buffer AVL to one tube of lyophilized carrier RNA. Dissolve Carrier RNA thoroughly. • Transfer to the buffer AVL bottle, and mix thoroughly before using buffer AVL for the first time. Lyophilized carrier RNA is stable for up to one year when stored at room temperature (+18°C/+25°C). Carrier RNA dissolved in buffer AVL, however, should be stored at +2°C/+8°C and will be stable for up to six months. If buffer AVL/carrier RNA is stored at room temperature, it will be stable for no more than two weeks. When stored at +2°C/+8°C, the buffer AVL/carrier RNA solutions forms a precipitate. This precipitate must be redissolved by warming at +80°C and the solution cooled to room temperature before use. NOTE: DO NOT warm buffer AVL/carrier RNA solution more than six times. DO NOT incubate at 80°C for more than 5 minutes. Frequent warming and extended incubation will cause degradation of carrier RNA, leading to reduced recovery of viral RNA and eventually false negative Rt-PCR results. This is particularly the case with low-titered samples. Also, it is recommanded to aliquot the buffer AVL/ARN.

6.1.2 AW1 buffer preparation • Buffer AW1 is supplied as a concentrate. Prior to first time use, add a volume of 25 mL of ethanol (96-100%) to the 19 mL of concentrated buffer. Buffer AW1 is stable for 1 year when stored closed at room temperature. 6.1.3 AW2 buffer preparation • Buffer AW2 is supplied as a concentrate. Prior to first use, add a volume of 30 mL of ethanol (96-100%) to the 13 mL of concentrated buffer. Buffer AW2 is stable for 1 year when stored closed at room temperature.

67-080B

6.2 Amplification kit

• All the reagents of this kit are ready to use following the instructions below.

• This kit must be stored at +2°C/+8°C until expiry date written on the box. D

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FOR RESEARCH USE ONLY

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67-080 - 05/09/01 - 2/8

4.

6.3.1 R21 Washing solution (10x) • If crystals are observed in the concentrated washing solution (R21), warm at +37°C until crystals dissolve before dilution of reagent. • Mix one volume of concentrated washing solution (10x) (R21) with 9 volumes of distilled water. • Prepare enough 1x solution for washing by filling all the wells ten times with 350 µL at each time. • Washing solution 1x may be stored 3 months at +2°C/+8°C. 6.3.2 R22 / R23 Conjugate (50x) • Dilute the conjugate 50x (R23) 1/50 in the conjugate diluent (R22) previously homogenized. • Mix 1 volume of concentrated conjugate (50x) (R23) with 49 volumes of conjugate diluent (R22). Prepare enough ready-to-use conjugate in order to dispense 100 µL per well. Example for 1 mL of diluted conjugate:

Mix 20 µL of R23 with 980 µL of R22.

Ready-to-use conjugate must be prepared just prior to use during the hybridization step. DO NOT store diluted conjugate. Precautions for the use of the kit WARNING: detergent and/or high protein concentrations in the extraction medium could interfere with the DNA coating in the microtiter plate. For this reason, it is important to follow the provided protocol and to use only the recommended or provided reagents within the 3 kits.

7.

Warning and precautions

For research use only. Not for use in diagnostic procedures.

7.1 Warning and precautions for Molecular Biology • Amplification procedures require highly skilled technique to avoid risk of sample contamination. • Areas corresponding to the sample preparation, amplification, amplified product analysis or aliquot of amplification reagents must be separated. Never introduce an amplified product in reagent or sample preparation areas. • Samples used must be exclusively reserved for this analysis. Samples must be prepared under a laminar flow hood. Tubes from different specimens are never opened at the same time. Pipettes used to handle samples are reserved for this purpose only. These pipettes are positive displacement pipettes or pipettes equiped with filter tips. The different types of tips used must be sterile. • The pipettes used to aliquot reagents must be reserved only for this purpose. The necessary reagents for amplification are aliquoted in order to be used during one single experiment.

7.2 General warning and precautions • Handle and dispose all specimens as if they contain infectious agents. • After use: material, reagents and waste must be handled as potentially infectious. • Read all instructions before performing this assay. • Do not use reagents after expiry date printed on the labels. • Use only reagents provided in the kit. • Do not interchange reagents from kits with different batch numbers or from other manufacturers. • Do not smoke, eat or drink in dedicated work areas.

7.3 Reagent specific warning and precautions • Buffer AVL and AW1 contains chaotropic salt which is an irritant. Take appropriate laboratory safety measures and wear gloves when handling. This component must not be used with disinfecting agents that contain bleach. Buffer AW2 and protease diluent contain 0.04% sodium azide as preservative. • Reagents R11 and R12 contains ß-mercaptoethanol: R23/24/25: Toxic by inhalation, skin contact or ingestion. S45-36/37/39: In case of an accident or if you feel ill, seek medical attention (show the label if possible). Wear suitable protective closing, gloves and eyes/face protection. • Denaturation solution 1 R15 contains EDTA: R36/37/38: Irritating to eyes, respiratory system and skin. S 26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S 46: If swallowed, seek medical advice immediately and show this container or label. • Denaturation solution 2 R16 contains sodium hydroxide: R36/38: Irritant for skin and eyes. S26-37/39-45: In case of contact with eyes rinse immediately with plenty of water and seek medical advice. Wear suitable gloves and eye/face protection. In case of accident or if you feel ill, seek medical advice immediately (show the label if possible). • Ready to use probes R19 and R20 contain Formamide: R61: May cause harm to the unborn child. R41: Risk of serious damage to eyes. R37/38: Irritating to respiratory system and skin. R45: In case of accident or if you feel unwell, seek medical advice immediately (show the label if possible). S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. S36/37/39: Wear suitable protective clothing, gloves and eye/face protection. S23: Do not breathe vapour. • Conjugate diluant R22, wash solution R21 contain 0.01% Thimerosal as preservative. • Avoid contact between the reagents and the skin. Wash immediately with copious amount of water if contact accurs. Wear gloves when handling the reagents.

8.

Sample treatment and transport

9.

Controls

The inhibition controls follow this principle: ENTERO

ENTEROVIRUS

ENTERO

ENTEROVIRUS AMPLIFIED FRAGMENT

ENTERO

ENTEROVIRUS POSITIVE CONTROL AMPLIFIED FRAGMENT

Identical LENTGH and GC % ENTERO

PLASMID

The use of ENTEROVIRUS or control probe allows the differentiation of positive sample from positive control

CLONING VECTOR • Use of controls is imperative to validate the manipulation.

9.1 Positive controls • A positive control may be added using infected culture cells by a laboratory Enterovirus strain. This control will be considered as a sample and will go through the complete extraction to detection process. • The kit contains a positive plasmid control (see above) which checks the capacity for Enterovirus consensus primers to amplify a fragment from a sequence corresponding to a Coxsackievirus B4. The amplification positive control is performed by using the inhibition control premix (R12) with 10 µL of water reverse transcription product. The plasmid included in the inhibition control premix is the DNA control and will go through the genomic amplification to detection process. The amplified product is then detected with the control probe (R20) provided in the detection kit (67-080C).

9.2 Inhibition controls • This control allows detection of amplification inhibitors in the sample. • The inhibition control premix is ready to use. Each sample will be amplified both with amplification premix (R11) and the inhibition control (R12).

9.3 Amplification negative control • This control proves the absence of contamination. It is performed with all the reagents except the sample (replaced by sterile, RNase free water) from the reverse transcription to the microplate detection. This control is tested in duplicate during the detection step.

9.4 Detection negative control (R14) • CSF is obtained following classical conditions of lumbar puncture. Nasopharyngeal secretions, throat swabs, and stools specimens are collected following standard laboratory protocols. • Treat all samples as potentially infectious. • Samples which are not treated upon arrival must be stored frozen at -80°C or below this temperature. • For samples to be transported, check your local legislation for hazardous and infectious material transport. • Nasopharyngeal secretions, throat swabs, and stools specimens must be kept frozen in transport medium and must be centrifuged at 4000-6000rpm 6 min. before extraction and analysis. CSF will be directly extracted.

ＣＯＳＭＯ ＢＩＯ ＣＯ.,ＬＴＤ.

• This control (R14) is provided (ready-to-use) for the cut-off determination. • This control is tested in duplicate during the detection step.

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PARC TECHNOLOGIQUE DELTA SUD 09120 VARILHES FRANCE TELEPHONE : 33 (0) 5 61 69 61 00 FAX : 33 (0) 5 61 69 61 01

FOR RESEARCH USE ONLY

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67-080 - 05/09/01 - 3/8

67-080C

6.3 Detection kit

3

10.3 Washing step

Sample extraction kit, QIAamp® Viral RNA Mini Kit

67-080A

In the room reserved for sample extraction • Equilibrate samples to room temperature at +18°C/+25°C. • Equilibrate AVE buffer to room temperature at +18°C/+25°C. • Check that buffer AW1, AW2 and carrier RNA have been prepared according to the instructions given in 6 "Reagent reconstitution". Use one aliquot per run. • Redissolve any precipitate in buffer AVL/carrier RNA by heating if necessary, and cool to room temperature before use. NB: All centrifugation steps are carried out at room temperature.

• Carefully open the spin column and add 500 µL of buffer AW1. Close the cap and centrifuge at 6000xg for one minute. Place the spin column in a clean 2 mL collection tube (provided), and discard the tube containing the filtrate. It is not necessary to increase the volume of buffer AW1 even if the original sample volume was larger than 140 µL. • Carefully open the spin column and add 500 µL of buffer AW2. Close the cap and centrifuge at full speed using a microcentrifuge for 3 minutes. Place the spin column in a clean 1.5 mL microcentrifuge tube (not provided) and discard the old collection tube with the filtrate. NOTE: Residual buffer AW2 in the eluate may cause problems in downstream applications. Some centrifuge rotors may vibrate upon deceleration, resulting in flow-through containing buffer AW2 to contact the spin column. Removing spin column and collection tube from the rotor may also cause flow-through to come into contact with the spin column. In these cases, the optional following step may be performed. Place the spin column in a new 2 mL collection tube (not provided) and discard the old collection tube with the filtrate. Centrifuge at full speed using a microcentrifuge for one minute.

10.1 Lysis step • Prepare and identify (on the lid) an equal number of 1.5 mL microcentrifuge tubes to samples being analyzed. • Pipet 560 µL of prepared AVL/RNA carrier in each tube. • Add 140 µL sample and mix by pulse-vortexing for 15 seconds. To ensure efficient lysis, it is essential that the sample is mixed thoroughly to yield a homogeneous solution. If the sample volume is larger than 140 µL, increase the amount of buffer AVL/carrier RNA proportionally (add 4 volumes of buffer for one volume of sample). If the volume is less than 140 µL, add sterile PBS buffer to adjust final volume to 140 µL. • Incubate at room temperature (+18°C/+25°C) for 10 minutes. Viral particle lysis is complete after 10 minutes at room temperature. Longer incubation times have no effect on the yield or quality of the purified RNA. Potentially infectious agents and RNases are inactivated in buffer AVL. • Briefly centrifuge the 1.5 mL microcentrifuge tube to remove any droplets from the inside of the lid.

Reverse transcription and amplification protocol 67-080B

Amplification kit

NOTE: Inhibition control premix (R12) contains DNA and should be stored at -18°C/-22°C in the extraction room upon receipt and manipulated ONLY in this room. • Prepare the Thermal Cycler. For each run of reverse transcription and amplification, identify the following numbers of tubes: - 3 tubes for each sample to be tested (reverse transcription premix, amplification premix and inhibition control premix); - 1 tube for the reverse transcription negative control; (all the reverse transcription reagents excepted the sample replaced by sterile water). - 1 tube for the positive control; - 1 tube for the amplification negative control.

10.4 Elution step • For CSF samples carefully open the spin column and add 40 µL of buffer AVE equilibrated to room tempertature. Close the cap and incubate for one minute at room temperature. Centrifuge at 6000xg for one minute. Place this eluate on the column, incubate one minute at room temperature and centrifuge at 6000xg for one minute. • For other samples the elution step needs to be performed only once using a volume of 60 µL. Close the cap and incubate for one minute at room temperature. Centrifuge at 6000xg for one minute. • Extracted viral RNA is stored at -70°C.

n = number of samples + 1 control tube n = number of necessary tubes for reverse transcription n = number of necessary tubes for the amplification premix n = number of necessary tubes for the inhibition premix

Example for 5 samples to be analyzed in one run: n = 5 + 1 = 6

In the room reserved for reagents preparation 11.1 Reverse trancriptase dilution preparation (R9) Prepare a reverse transcriptase and RNase inhibitor dilution in RNase free water for (n+1) tubes: • Pipet (n+1) x 1 µL of transcriptase Omniscript™ (R9) in a tube. • Add (n+1) x 0.25 µL of RNase inhibitor (R10). • Add (n+1) x 2 µL of RNase free water.

10.2 Loading step • Add 560 µL of 96-100% ethanol to the sample, and mix by pulsevortexing for 15 seconds. Only ethanol should be used since other alcohols may result in reduced RNA yield and purity. If the sample volume is larger than 140 µL, increase the amount of ethanol proportionally (add 4 volumes of ethanol for one volume of sample). • After mixing, briefly centrifuge the 1.5 mL microcentrifuge tube to remove any droplets from inside of the lid. • Prepare and identify the same number of spin columns as samples to be tested. Carefully apply 630 µL of the sample to the spin column (in a 2 mL collection tube) without wetting the rim. Close the cap, and centrifuge at 6000xg for 1 minute. • Place the spin column into a clean 2 mL collection tube, and discard the tube containing the filtrate. Carefully open the spin column and repeat preceding step. If the sample volume was larger than 140 µL, repeat this step until all of the lysate has been loaded onto the spin column.

4

11.

Example for 5 samples (n = 6):

Pipet 7 µL of R9 + 1.75 µL of R10 + 14 µL of RNase free water in a tube.

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PARC TECHNOLOGIQUE DELTA SUD 09120 VARILHES FRANCE TELEPHONE : 33 (0) 5 61 69 61 00 FAX : 33 (0) 5 61 69 61 01

ＣＯＳＭＯ ＢＩＯ ＣＯ.,ＬＴＤ.

FOR RESEARCH USE ONLY

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67-080 - 05/09/01 - 4/8

10.

Sample extraction protocol

• Distribute 7 µL of reverse transcription premix ready-to-use (R8) in each of the n corresponding tubes. • Add one drop (40-50 µL) of mineral oil in each of the tubes. Example for 5 samples (n = 6):

Distribute 7 µ L in each of the six tubes reverse transcription premix.

11.6 Inhibition control premix preparation (R12) • Pipet (n+1) x 35 µL inhibition control premix (R12) and dispense it in a 1.5-2 mL tube. • Add (n+1) x 5 µL of diluted HotStarTaq™. • Distribute 40 µL in each of the n tube.

12.

Example for 5 samples (n = 6):

Detection kit,

11.3 Diluted HotStarTaq™ preparation WARNING: to obtain optimal performance of the kit, you must use the polymerase recommended in the procedure. Use the HotStarTaq™ QIAGEN Ref.: 203203 (250 U) 5 Units/µL • From the concentrated HotStarTaq™, prepare a dilution in sterile water for 2x (n+1) tubes in order to get the necessary quantity for the amplification premix tubes, inhibition control premix tubes and control tubes: • Pipet 2 x (n + 1) x 5 µL of sterile water in one tube. • Add 2 x (n + 1) x 0.3 µL of HotStarTaq™ at 5 units/µL. Example for 5 samples (n = 6):

Pipet 70 µL of sterile water and add 4.2 µL of concentrated HotStarTaq™.

11.4 Amplification premix preparation (R11) • Pipet (n+1) x 35 µL of amplification premix (R11) and dispense it in a 1.5-2 mL tube. • Add (n+1) x 5 µL of diluted HotStarTaq™. • Distribute 40 µL in each of the n tubes. Example for 5 samples (n = 6):

Pipete 245 µL of R11 and add 35 µL of diluted HotStarTaq™. Distribute 40 µL in each of the six amplification premix tubes.

• Place the prepared amplification premix tubes and the rest of diluted HotStarTaq™ on ice. • Carefully close all the tubes before moving to the new location. • Take the diluted reverse transcriptase, the diluted HotStarTaq™ (volume dedicated to the inhibition control tubes) as well as all the prepared premix tubes and n empty tubes for the inhibition control premix, and go to the room reserved for the sample extractions.

In the room reserved for sample extraction 11.5 Reverse transcription • Keep the amplification premix tubes and the diluted HotStarTaq™ on ice during reverse transcription. • For each sample add 10 µL of extracted sample in reverse transcription premix tube through the oil. • Add 10 µL of water for the control in an other reverse transcription premix tube through the oil (tube identified "reverse transcription negative control"). Example for 5 samples:

For each extracted sample, add 10 µL in each tube of reverse transcription premix tube. Add 10 µ L of water in the 6 th reverse transcription premix tube.

• Denature 10 min. at +60°C. • Bring back temperature to +37°C. • Add 3 µL of diluted reverse transcriptase per tube through oil. • Incubate 45 min. at +37°C. • Stop the reaction by incubation 5 min. at +90°C. • The obtained cDNA are immediately treated for amplification or stored at -18°C/-22°C.

Pipet 245 µL of R12 and add 35 µL of diluted HotStarTaq™. Distribute 40 µL in each of the 6 inhibition control premix.

Detection protocol 67-080C

For each run, select a well for each amplified product and wells for each control following this model:

11.7 Addition of samples and controls in premix solutions • Sample well: Add the transcription product obtained from sample in the following - 1 well each amplified sample to hybridize with generic Enterovirus probe (R19) premix: - 1 well each corresponding inhibition control, to hybridize with control probe (R20) • 10 µL of sample reverse transcription product in 1 prepared • Control well for each run: amplification premix tube, properly identified. • 10 µL of sample reverse transcription product in 1 prepared - 1 well amplified positive control to hybridize with control probe (R20) inhibition control premix tube, properly identified. - 1 well amplification negative control to hybridize with Enterovirus probe (R19)

Add the transcription product obtained without sample in the following - 1 well amplification negative control " " control probe (R20) premix: 1 well detection negative control (R14) " " Enterovirus probe (R19) • 10 µL of water reverse transcription product in 1 prepared amplification premix tube for the preparation of amplification negative - 1 well detection negative control (R14) " " control probe (R20) control. Plan 15 wells on the microplate: • 10 µL of water reverse transcription product premix in 1 prepared Example for 5 samples: - 5 amplified sample wells inhibition control premix tube for the amplification positive control. - 5 inhibition control wells - 5 control wells following description above. Note that the water reverse transcription product allows performing both positive and negative amplification controls. • Draw a microplate map.

In the amplification room • Remove the tubes from the thermocycler. • Add one drop of mineral oil in each of the tubes. • Run the following amplification program. NOTE: the 15 min. at +94°C denaturation step is imperative. This allows the DNA polymerase (HotStarTaq™ QIAGEN) activation. NOTE: volume in each tube is 50 µL.

Amplification program

In the room reserved for the post-amplification 12.1 Denaturation and fixation

Thermocycler w/o heating lid

with heating lid

Perkin Elmer 480 for ex

Perkin Elmer 9600 or 2400 / 9700

1 Cycle

94°C

15 min.

94°C

15 min.

5 Cycles

94°C 52°C

15 sec. 2 min.

94°C 52°C

15 sec. 2 min.

Ramp 3 sec./°C or 40%

35 Cycles

• R14 is the negative microplate control ready-to-use, provided in the kit for the calculation of cut-off value. This control is tested in duplicate and must be treated like the other amplified products. • The various other controls should be treated like the other amplified products.

72°C

1 min. 15 sec.

72°C

1 min. 15 sec.

94°C 54°C

15 sec. 50 sec.

94°C 54°C

15 sec. 1 min. 15 sec.

74°C

50 sec.

74°C

50 sec.

+4°C

∞

+4°C

∞

• For each sample and control, distribute 5 µL of the amplified product in 1.5-2 mL new polypropylene identified tubes. • Add 10 µL of denaturation solution 1 (R15) and vortex. Add 10 µL of denaturation solution 2 (R16), vortex and incubate for 5 min. at room temperature. • Add 200 µL of coating solution (R17) and vortex. WARNING: carefully identify the microplate wells (R18) in order to facilitate the interpretation of results.

Ramp 3 sec./°C or 40%

• The amplification products may be analyzed immediately or may be stored frozen at -18°C/-22°C to be analyzed later. • For long term storage (>1 month) add 1 µL EDTA in each amplified product and store at -18°C/-22°C.

ＣＯＳＭＯ ＢＩＯ ＣＯ.,ＬＴＤ.

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PARC TECHNOLOGIQUE DELTA SUD 09120 VARILHES FRANCE TELEPHONE : 33 (0) 5 61 69 61 00 FAX : 33 (0) 5 61 69 61 01

FOR RESEARCH USE ONLY

t

67-080 - 05/09/01 - 5/8

11.2 Reverse transcription premix preparation (R8)

5

12.2 Hybridization • Warm the ready-to-use Enterovirus generic probe (R19) (red) and the control probe (R20) (blue) 15 min. at +37°C prior to use. • Empty the wells by overturning the plate. • Distribute (per well) 100 µL of Enterovirus generic probe (R19) (red): - in the samples wells; - in one of the two negative amplification control wells; - in one of the two negative detection control wells. • Distribute (per well) 100 µL of control probe (R20) (blue): - in the inhibition control wells; - in the positive control well; - in the other negative detection control well; - in the other negative amplification control well. • Cover with an adhesive (R26) and incubate 30 min. at +37°C.

12.3 Post hybridization washings • Reconstitute the 10x washing solution (R21) as described in section 6 "Reagents reconstitution". • Wash the plate with a microplate washer or manually: • For automated washing: a/ Program a 500 µ L purge before beginning the wash procedure to clear the lines of the wash fluid; b/ Aspirate contents of wells; c/ Fill the wells with 350 µL of 1x washing solution. Soak for 30 seconds and aspirate dry (check for complete aspiration); d/ Repeat step “ c ” 4 additional times; e/ Tap the plate dry on paper towels to eliminate residual washing solution. • For manual washing: a/ Empty the wells by overturning and tap dry on paper towels; b/ Dispense 350 µL of 1x solution with a repeater pipette or a wash bottle. Soak for 30 seconds. Empty the wells by overturning and tap dry on paper towels; c/ Repeat step “ b ” 4 additional times.

12.4 Detection and post-detection washings • Before the end of hybridization step: allow the conjugate diluent (R22) to warm up to room temperature (+18°C/+25°C). Dilute the conjugate 50x (R23) to 1/50 in the diluent, according the instruction given in section 6 "Reagent reconstitution"; • At the end of the hybridization step, empty the wells by inverting the plate. Add 100 µL of ready-to-use conjugate by using a repeater pipette or other suitable pipetting device; • Incubate 15 min. at room temperature; • Wash as described in "12.3".

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12.5 Revelation • Allow the substrate diluent (R24) to warm up to room temperature. Prepare in an amber vial (or clear glass vial covered with aluminium) the total volume of substrate (R24) needed (100 µL per well); • Empty the wells by inversion. • Dispense 100 µL of substrate for each well; • Incubate 30 min.at room temperature in the dark; • Stop the reaction by adding 100 µL of stop solution (R25) to each well; • Blank the plate reader against air and read the optical density (OD) at 450 nm. A double reading may also be performed (450/650 nm).

13.

Calculation and results interpretation

13.1 Validation of the test and cut-off value determination • The test interpretation for each run is possible only if the OD obtained with the detection negative controls (both hybridized with Enterovirus generic probe and control probe) is < 0.4 OD for each of the two corresponding wells. • Cut-off determination: A cut-off value (CO) is calculated from the mean of the two negative detection control values following hybridization with each of the two probes. • Calculation is done as follows: - if OD reading is 450 nm: CO = OD mean + 0.2 - if OD reading is 450/650 nm: CO = OD mean + 0.10 • Make sure that the amplification is not contaminated: the OD obtained with amplification negative controls (H2O) must be below the cut-off value. • The OD obtained with the amplification positive controls must be above the cut-off value. If one of these conditions is not met, the technique used in the test should be considered suspect and the test repeated. • The obtained results are then analyzed.

Before finally validating a negative result it is necessary to analyze these results with its corresponding inhibition control result: • If the inhibition control is >0.8 OD, the sample can be amplifed and result with Enterovirus probe is confirmed negative. • If the inhibition control is CO + 10%; Amplification is positive for Enteroviruses. If OD sample < CO - 10%; Amplification is negative for Enteroviruses. If OD sample = CO ± 10%; Amplification is indeterminant and amplified product must be retested. • Following retesting of an indeterminant amplified product: If OD sample > CO + 10%; Amplification is positive for Enteroviruses. If OD sample < CO - 10%; Amplification is negative for Enteroviruses. If OD sample = CO ± 10%; Obtain a new sample and retest.

ＣＯＳＭＯ ＢＩＯ ＣＯ.,ＬＴＤ.

Possibles causes: • Washing problem.

• Conjugate concentration is too high. • Problem during conjugate deposit. • Incubation temperature of conjugate is too high.

Solutions: • Follow the protocol, time and number of the post hybridization and post detection washes. • Check conjugate dilution. • Conjugate should be pipetted at the bottom of the well, not at the top. • Check the room temperature (+18°C/+25°C).

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FOR RESEARCH USE ONLY

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• Distribute 100 µL per well following this model: - in one well for each sample; - in one well for each amplified sample with the inhibition control; - in one well for the positive control; - in 2 wells for the negative amplification control; - in 2 wells for the negative detection control. • Cover the plate with an adhesive (R26) and incubate for 1 hour at +37°C. Alternatively, you may incubate overnight at room temperature.

15.

Performances of the test

15.1 Analysis of sample from the Second QCCA Enterovirus Proficiency Panel - April 2000. Analysis of these samples showed: • 8/9 Enterovirus positive samples were detected. The sample containing 0.39 DCIT of Cox A9 gave an OD reading of 1.3. The sample containing 0.039 DCIT of Cox A9 was not detected. • Sample containing Rhinovirus 3 was found positive. • Other sample gave the expected negative result.

16.

References

(1) BARRANGER C., KABACHE N., CRÉTÉ N., JOANNÈS M. Enterovirus Detection through Consensus Amplification and microplate hybridization. (Poster). European Society of Clinical Virology, Ghent, Belgium, January 2001. (2) BARRANGER C., KABACHE N., CORVAISIER C., JOANNÈS M. Enterovirus Consensus Amplification and Detection. (Poster). American Society of Microbiology, Orlando, USA, May 2001.

15.2 Reference strains

Serotype

Strain

Serotype

Strain

Serotype

Strain

Serotype

Strain

Serotype

Strain

Serotype

Strain

Serotype

Strain

Serotype

Strain

PV-1

Mahoney

CV-A6

Gdula

CV-A14 a

G-14

CV-A22

Chulman

E-1 a

Farouk

E-11

Gregory

E-19

Burke

E-30

Bastianni

PV-2

Lansing

CV-A7

Parker

CV-A15

G-9

CV-A24

Joseph

E-2

Cornelis

E-12

Travis

E-20 a

JV-1

E-31

Caldwell

PV-3

Leon

CV-A8

Donovan

CV-A16

G-10

CV- B1

Conn-5

E-3 a

Morrisey

E-13 a

Del Carmen

E-21 a

Farina

E-32

PR-10

CV-A1

Tompkins

CV-A9 a

Bozek

CV-A17

G-12

CV- B2

Ohio-1

E-4

Pesascek

E-14

Tow

E-24

DeCamp

E-33

Toluca-3

CV-A2

Fleetwood

CV-A10

Kowalik

CV-A18

G-13

CV- B3

Nancy

E-5 a

Noyce

E-15

Ch 96-51

E-25

JV-4

EV-68

Fermon

CV-A3

Olson

CV-A11 a

Belgium-1

CV-A19

Dohi

CV- B4

JVB

E-6 a

D'Amori

E-16

Harrington

E-26

Coronel

EV-69

Toluca-1

CV-A4

High Point

CV-A12 a

Texas-12

CV-A20

IH-35

CV- B5

Faulkner

E-7

Wallace

E-17

CHHE-29

E-27

Bacon

EV-70

J 670/71

CV-A5

Swartz

CV-A13 a

Flores

CV-A21

Coe

CV- B6

Schmitt

E-9

Hill

E-18

Metcalf

E-29

JV-10

EV-71

BrCr

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• 64 reference strains were analyzed with the kit; all of them gave positive result. (Colimon R., Caro V., Bourlet T., Minjolle S., Jusselin I., Pozzeto B. and Crainic R.; 2000 - J. Clin. Virol. 18 - 1-3:163).

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17.

Outlined procedure

ENTEROVIRUS CONSENSUS ref.: 67-080

Sample extraction protocol

Reverse transcription and amplification protocol

Detection protocol

For each run of reverse transcription and amplification, identify the following numbers of tubes: - 3 tubes for each sample to be tested (reverse transcription premix, amplification premix and inhibition control premix). - 1 tube for the reverse transcription negative control (all the reverse transcription reagents excepted the sample replaced by sterile water). - 1 tube for the positive control - 1 tube for the amplification negative control

Select a well for each amplified product and wells for each control following:

n = number of samples + 1 control tube STEPS

LYSIS

REAGENTS

INCUBATION

Add 560 µL ethanol. Place spin column on collection tube. Add 630 µL sample. Repeat this step.

WASHING

Open the column. Add 500 µL AW1. Change tube. Add 500 µL AW2. Place spin column on a 1.5 mL tube.

ELUTION

REAGENTS

INCUBATION

STEPS

Others: Add 60 µL AVE.

INCUBATION

DILUTE REVERSE TRANSCRIPTASE

Pipet (n + 1) x 1 µL R9. Add (n + 1) x 0.25 µL R10. Add (n + 1) x 2 µL water RNase free.

DENATURATION

Take 5 µL sample or control. Add 10 µL R15. Add 10 µL R16.

Centrifuge 6000xg. 1 min.

REVERSE TRANSCRIPTION PREMIX

Distribute 7 µL R8. Add a drop of mineral oil.

FIXATION

Add 200 µL R17 Distribute 100 µL per well.

Vortex. Incubate 1 h. at +37°C. or overnight at RT°.

HYBRIDIZATION

Warm probes R19 and R20 Empty the wells. Distribute 100 µL probe.

15 min. at +37°C.

WASHING

Aspirate contents of wells. Fill 350 µL of R21 1X. Repeat 4 times.

REVELATION

Empty the wells. Distribute 100 µL R23 at 1/50 diluted with R22.

WASHING

Aspirate contents of wells. Fill 350 µL of R21 1X. Repeat 4 times.

REVELATION

Empty the wells. Distribute 100 µL R24.

STOP

Distribute 100 µL R25.

READING

OD at 450 nm or 450/650 nm.

Centrifuge 20000xg. 3 min.

CSF: Add 40 µL AVE.

REAGENTS

In the post-amplification reserved room

Vortex 15 sec. Incubate 10 min. at RT°. Centrifuge briefly. Vortex 15 sec. Centrifuge briefly.

Centrifuge 6000xg. 1 min.

Eluate again on the column.

1 min. at RT°. Centrifuge 6000xg. 1 min. Incubate 1 min. at RT°. Centrifuge 6000xg. 1 min. Incubate 1 min. at RT°. Centrifuge 6000xg. 1 min.

Pipet 2 x (n + 1) x 5 µL sterile H2O. Add 2 x (n + 1) x 0.3 µL HotStarTaq™.

DILUTED HotStarTaq™

Pipet (n + 1) x 35 µL R11. Add (n + 1) x 5 µL diluted HotStarTaq™. Dispense 40 µL.

AMPLIFICATION PREMIX

In the samples extraction room

REVERSE TRANSCRIPTION

Samples can be stored frozen at -70°C

Add 10 µL sample or water in reverse transcription premix. Add 3 µL diluted reverse transcriptase.

Denature 10 min. at +60°C. Bring back to +37°C. Incubate 45 min. at +37°C. Stop: 5 min. at +90°C.

INHIBITION CONTROL PREMIX

Pipet (n + 1) x 35 µL R12. Add (n + 1) x 5 µL diluted HotStarTaq™. Dispense 40 µL.

ADDITION of SAMPLES and CONTROLS

10 µL cDNA sample in amplification premix. 10 µL cDNA sample in inhibition control premix. 10 µL water reverse transcription product in amplification premix. 10 µL water reverse transcription product inhibition control premix.

In the amplification reserved room

AMPLIFICATION

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• Control well for each run: - 1 well amplified positive control to hybridize with control probe (R20) - 1 well amplification negative control to hybridize with Enterovirus probe (R19) - 1 well amplification negative control " " control probe (R20) - 1 well detection negative control (R14) " " Enterovirus probe (R19) - 1 well detection negative control (R14) " " control probe (R20)

In the reagents preparation room

Pipet 560 µL AVL/ARN. Add 140 µL sample.

LOADING

STEPS

• Sample well: - 1 well each amplified sample to hybridize with generic Enterovirus probe (R19) - 1 well each corresponding inhibition control, to hybridize with control probe (R20)

Vortex. Vortex. Incubate 5 min. at RT°.

Incubate 30 min. at +37°C. Incubate 30 sec. at RT°.

Incubate 15 min. at RT°. Incubate 30 sec. at RT°.

Incubate 30 min. at RT°. in a dark location.

INTERPRETATION

Run the amplification program. After amplification, immediately analyze the products of amplification or store at -18°C/-22°C by adding 1 µL EDTA to each amplified product.

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