Procedure: illumigene® hsv 1&2 Procedure and revision number: sn11107 Rev. 07/15 Meridian Catalog #280650




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Procedure: illumigene® HSV 1&2
Procedure and revision number: SN11107 Rev. 07/15
Meridian Catalog #280650



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NOTE: This procedure is provided to Meridian’s customers to assist with the development of laboratory procedures. This document was derived from, and was current with, the instructions for use (IFU) that accompanies the product at the time it was created. The user is instructed to consult the IFU packaged with the product to ensure currency of the procedure prior to adapting the document to routine laboratory use and periodically thereafter to ensure future IFU modifications, which might affect this procedure, are identified. Any modifications to this document are the sole responsibility of the person making the modifications.

PRINCIPLE:

The illumigene HSV 1&2 DNA amplification assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection and differentiation of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) in cutaneous and mucocutaneous lesion specimens from male and female patients suspected of Herpetic infections.


illumigene HSV 1&2 utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect HSV-1 and HSV-2 by targeting segments of the herpes simplex virus 1 and herpes simplex virus 2 genomes. The assay individually targets regions of the HSV-1 and HSV-2 genomes in two separate Test Devices. The HSV-1 target is a 208 base pair (bp) sequence of the HSV-1 glycoprotein G (US4) gene. The HSV-2 target is a 189 bp sequence of the HSV-2 glycoprotein G (US4) gene. Results from illumigene HSV 1&2 are used as an aid in the diagnosis of HSV infection in symptomatic patients.
The assay is intended for use in hospital, reference or state laboratory settings. This device is not intended for nonlaboratory point-of-care use.
WARNING: illumigene HSV 1&2 is not intended to be used with cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV infections of the central nervous system (CNS). The device is not intended for prenatal screening.

SPECIMEN:

Preferred Sample Types:

Human cutaneous and mucocutaneous lesion swab samples in viral transport media. The following viral transport media are acceptable for use: MicroTest™ M4®, MicroTest™ M4RT®, MicroTest™ M6™, UniTranz-RT™ Transport System, Copan UTM™ Virus Collection, HealthLink® UTM or identical transport medium*, BD Universal Viral Transport Medium (UVT), ViraTrans™ VTM, Hardy Diagnostics VTM.



*HealthLink UTM is equivalent to Quest V-C-M Medium
Undesirable samples:

1. Swab samples in viral transport media with protein stabilizers.

2. Calcium alginate swab samples.
Collection and Storage:

Cutaneous and mucocutaneous lesion sample collection should be performed in accordance with institutional guidelines for collection of clinical specimens for herpes simplex virus infection.


Place swab(s) in a viral transport medium. Samples should be stored refrigerated (2-8 C) after collection and during transportation to the laboratory. Samples should be tested as soon as possible, but may be stored refrigerated (2-8 C) up to 7 days prior to testing. Do not freeze samples. Do not store at room temperature.

This facility’s procedure for specimen collection is: _____________________________________ ______________________________________________________________________________

This facility’s procedure for transporting specimens is: __________________________________ ______________________________________________________________________________

This facility’s procedure for rejected specimens is: _____________________________________ ______________________________________________________________________________



MATERIALS AND EQUIPMENT:

MATERIALS PROVIDED:

  1. illumigene Sample Preparation Apparatus III (SMP PREP III): Tris-buffered solution containing formalin-treated E. coli harboring S. aureus DNA and sodium azide (0.09%) as a preservative.

  2. illumigene HSV 1 Test Device: Two-chambered device containing lyophilized amplification reagents (DNA polymerase, deoxynucleotide triphosphates) and either HSV-1-specific primers (TEST chamber) or S. aureus-specific primers (CONTROL chamber).

  3. illumigene HSV 2 Test Device: Two-chambered device containing lyophilized amplification reagents (DNA polymerase, deoxynucleotide triphosphates) and either HSV-2-specific primers (TEST chamber) or S. aureus-specific primers (CONTROL chamber). The illumigene HSV 2 Test Device is visually identified by an orange band on the Test Device closure tab.

  4. Mineral Oil (bottle with dropper tip)

  5. illumigene Heat Treatment (HT) Tubes

  6. Transfer Pipettes


MATERIALS NOT PROVIDED:

MATERIALS:

  1. illumigene HSV 1&2 External Control Kit, Catalog Number: 279960

  2. Disposable latex gloves, powder free

  3. DNase/RNase-free, aerosol resistant pipette tips

  4. Viral transport medium: MicroTest™ M4®, MicroTest™ M4RT®, MicroTest™ M6™, UniTranz-RT™ Transport System, Copan UTM™ Virus Collection, HealthLink® UTM or identical transport medium*, BD Universal Viral Transport Medium (UVT), ViraTrans™ VTM, Hardy Diagnostics VTM.

*HealthLink UTM is equivalent to Quest V-C-M Medium

EQUIPMENT NOT PROVIDED:

  1. Dry-bath with 12 mm heat block capable of 95 C

  2. Digital thermometer with Max/Min Temperature Memory (eg, Traceable® Lollipop™ Waterproof/Shockproof Thermometer)

  3. Vortex mixer

  4. Interval timer

  5. Micropipette capable of dispensing 50 μL

  6. iIlumipro-10, Meridian Bioscience, Inc. Catalog Number: 610172


REAGENT PREPARATION:

Ensure kit reagents are at room temperature (19-30 C) before use. Incorrect results may be obtained if reagents are not brought to room temperature prior to use.


PRECAUTIONS:

  1. All reagents are for in vitro diagnostic use only.

  2. Viral transport media containing protein stabilizers, such as MicroTest™ M5® and calcium alginate swabs, are not acceptable for illumigene HSV 1&2 specimen collection and transport.

  3. Do not interchange the Sample Preparation Apparatus or Test Devices between lots. Mineral Oil and Heat Treatment Tubes are interchangeable provided they are within the assigned expiration date when used.

  4. Follow Biosafety Level 2 and Good Laboratory practices during testing.9 Treat all specimens and used Test Devices as capable of transmitting infectious agents. Do not eat, drink or smoke in areas where specimens or kit reagents are handled.

  5. Wear disposable gloves while handling specimens and thoroughly wash hands afterwards.

  6. Quality Control Programs for Molecular Testing Laboratories, including proper use and care of equipment, should be employed.10

  7. The illumigene HSV 1&2 Test Devices contain lyophilized reagents. The protective pouches should not be opened until ready to perform the assay.

  8. The illumigene HSV 1&2 Test Devices include a latch feature that is designed to prevent contamination of the test area with amplification product. Do NOT use Test Devices with broken latches.

  9. Dispose of used illumigene HSV 1&2 Test Devices immediately after processing, leaving the device latch securely in place. Do NOT open the Test Device after processing. Opening the device after amplification may result in contamination of the test area with amplification product.

SHELF LIFE AND STORAGE:

The expiration date is indicated on the kit label. Store the kit at 2 - 30 C.


At this facility, kits are stored: _______________________________________________
CALIBRATION:

There are no calibrations associated with this procedure.


QUALITY CONTROL:

This test should be performed per applicable local, state, or federal regulations or accrediting agencies.

  1. Each device contains an internal control that controls for amplification inhibition, assay reagents and sample processing effectiveness. Internal control DNA is present in the SMP PREP III and is processed through all steps of the procedure. Primers for amplification of internal control DNA are present in the Control Chamber of the illumigene Test Device.

  2. The heat treatment step is monitored with an external thermometer and interval timer. Use of the max/min temperature memory of the thermometer to ensure that a temperature of 95 ± 5 C is maintained. Use of the interval timer to ensure that heat-treatment duration is 10 ± 2 minutes.

  3. Good laboratory practice recommends the use of control materials. Users should follow the appropriate federal, state and local guidelines concerning the running of external quality controls.

  4. illumigene HSV 1&2 External Control Reagents are supplied separately (Catalog 279960). It is recommended that the reactivity of each new lot and each new shipment of illumigene HSV 1&2 be verified on receipt and before use. External control tests should be performed thereafter in accordance with appropriate federal, state and local guidelines. The illumigene HSV 1&2 test kit should not be used in patient testing if the external controls do not produce the correct results.

  5. A separate Test Device must be used for each external control reagent.

QC Testing Frequency and Documentation:


For this facility, External QC is run: __________________________________________________
Results of External QC and action(s) taken when control results are unacceptable are documented: ___________________________________________________________________


PROCEDURE:

NOTE: Ensure that the illumipro-10 is powered on and required performance verifications have been completed prior to initiation of SPECIMEN PREPARATION. Refer to the illumipro-10 Operator’s Manual for further information regarding instrument set-up and operation.
NOTE: Ensure specimens are at room temperature (19-30 C) before specimen preparation.
SPECIMEN PREPARATION:


              1. Vortex the collected swab in transport medium for approximately 45-60 seconds.

              2. Using a transfer pipette, transfer 50 µL of the swab medium into one illumigene SMP PREP III. Discard the transfer pipette. Label the SMP PREP with the specimen identification.

              3. Replace SMP PREP III cap and vortex for approximately 10 seconds.

              4. Remove the tip cap from the SMP PREP III and squeeze the entire contents of the SMP PREP III into a Heat Treatment Tube. Label the tube with the specimen identification.

              5. Repeat SPECIMEN PREPARATION steps for all samples to be processed. Processed samples may be held up to 2 hours at room temperature (19-30 C) prior to heat treatment.

              6. Heat each Heat Treatment Tube containing sample in a dry-bath/heat block at 95 ± 5 C for 10 ± 2 minutes. Monitor heat-treatment step with digital thermometer and interval timer.

              7. Remove each Heat Treatment Tube from the dry-bath/heat block. Heat-treated samples may be held at room temperature (19-30 C) for up to 15 minutes prior to testing.


NOTE: The amount of sample prepared at the end of SPECIMEN PREPARATION is enough to complete both illumigene HSV 1 and HSV 2 testing.
TEST PROCEDURE:

NOTE: A maximum of 5 samples can be processed for both HSV 1 and HSV 2 in a single illumipro-10 run. HSV 2 Test Devices are identified by an orange band on the Test Device closure tab.


  1. Vortex heat-treated samples for approximately 10 seconds.

  2. Use 1 illumigene HSV 1 Test Device and 1 illumigene HSV 2 Test Device per sample. Remove the devices from their protective pouches. Carefully open the devices, holding the chambers such that the lyophilized reagents will not fall out upon opening. Place devices on a flat surface or in a rack that can accommodate the devices.

  3. Using a micropipette, transfer 50 µL of the sample to both the TEST (White Bead) and CONTROL (Yellow Bead) chambers of the illumigene HSV 1 Test Device and the HSV 2 Test Device. Take care to not introduce air to the reaction mixtures.

  4. Add 1 drop of Mineral Oil to both the TEST and CONTROL chambers of the illumigene HSV 1 and HSV 2 Test Devices. Close the illumigene Test Devices and fasten the latches securely.

  5. Tap each device on the bench top or mix to remove air bubbles. Carefully examine each Test Device for rehydration of the Control/Test Beads, for air bubbles left in the chamber and liquid in the top of the device. If undissolved beads, air bubbles or liquid in the top of the device are noted, tap the device on the bench top and repeat visual inspection. Amplification and detection should be initiated within 15 minutes.

  6. Repeat TEST PROCEDURE steps for all samples to be tested.

  7. Insert the illumigene Test Devices into the illumipro-10 and initiate run. Results will be displayed at the conclusion of the run.


INTERPRETATION OF RESULTS:

Sample ID

Reported Result

Interpretation

Patient Specimen

POSITIVE

HSV 1 TEST DEVICE: Sample contains HSV-1 target DNA.

HSV 2 TEST DEVICE: Sample contains HSV-2 target DNA.

NEGATIVE

HSV 1 TEST DEVICE: No HSV-1 target DNA detected.

HSV 2 TEST DEVICE: No HSV-2 target DNA detected.

INVALID

No reportable result. Repeat the test using the original sample.

Inhibitory patient specimen, improper sample preparation, reagent failure, instrument failure or internal control failure.



Positive Control

POSITIVE

Valid positive control result. Reagents active at time of use, illumipro-10 performing correctly.

NEGATIVE

Incorrect control result. Repeat the control tests as the first step in determining the root cause of the failure. If control failures are repeated please contact Meridian’s Technical Services Department at 1-800-343-3858 (US) or your local distributor.

INVALID

No reportable result. Repeat the control tests as the first step in determining the root cause of the failure. If control failures are repeated please contact Meridian’s Technical Services Department at 1-800-343-3858 (US) or your local distributor. Improper sample preparation, reagent failure, instrument failure or internal control failure.

Negative Control

POSITIVE

Incorrect control result. Repeat the control tests as the first step in determining the root cause of the failure. If control failures are repeated please contact Meridian’s Technical Services at 1-800-343-3858 (US) or your local distributor.

NEGATIVE

Valid negative control result. Reagents active at time of use, illumipro-10 performing correctly.

INVALID

No reportable result. Repeat the control tests as the first step in determining the root cause of the failure. If control failures are repeated please contact Meridian’s Technical Services Department at 1-800-343-3858 (US) or your local distributor. Improper sample preparation, reagent failure, instrument failure or internal control failure.

EMPTY WELL

NONE

No illumigene Test Device in the illumipro-10 Well.

OR

The illumigene Test Device present is compromised due to sample preparation failure, dirty device or improperly seated device. Repeat the test using original sample.




CALCULATIONS:

There are no calculations associated with this procedure.


REPORTING OF RESULTS:

Positive Test: Sample contains HSV-1 or HSV-2 target DNA

Negative Test: No HSV-1 or HSV-2 DNA detected
EXPECTED RESULTS:

The prevalence of HSV-1 and HSV-2 was calculated during 2014-2015 clinical studies based on the age of patient and by lesion anatomical location. The overall incidence during the study was 20.5% (237/1155) for HSV-1 and 19.4% (224/1156) for HSV-2.


LIMITATIONS OF THE PROCEDURE:

  1. Performance of illumigene HSV 1&2 has been established with prospective, male and female cutaneous and mucocutaneous lesion specimens only. Performance with other specimen types (frozen specimens or CSF) has not been established.

  2. The assay is not intended to be used for prenatal screening.

  3. Medications containing the active ingredient Zincum Gluconicum produced invalid results when tested at 7% v/v with illumigene HSV 1&2. The concentration of Zincum Gluconicum tested is excessive compared to the expected concentration of Zincum Gluconicum present in clinical samples; lower concentrations were not tested. The effect of Zincum Gluconicum on illumigene HSV 1&2 results have not been determined using clinical specimens.

  4. Casein at concentrations greater than 5 mg/mL was found to interfere with the illumigene HSV 1&2 assay.

  5. Due to confirmed unacceptable results, refrigerated storage (2-8 C) will not be considered an acceptable storage condition for SMP PREP III processed specimens to be tested with the illumigene HSV 1&2 DNA Amplification Assay.

  6. Negative results do not preclude infection and should not be used as the sole basis for treatment or other patient management decisions. This product can only be used with the illumipro-10 instrument.

  7. illumigene HSV 1&2 is a qualitative assay and does not provide quantitative values or information about organism load.

  8. The detection of nucleic acids is dependent upon proper specimen collection, handling, transportation, storage and preparation. Failure to observe proper procedure in any one of these steps can lead to incorrect results.

  9. Organism nucleic acid may persist in vivo, independent of organism viability. The illumigene HSV 1&2 does not distinguish between viable and nonviable organisms.

  10. As with all molecular based diagnostic tests, (A) False-negative results may occur from the presence of inhibitors, mutations or polymorphisms in the HSV target region, technical error, sample mix-up or low numbers of organisms in the clinical specimen; (B) False-positive results may occur from the presence of cross-contamination by target organisms, their nucleic acids or amplified product, and from nonspecific signals.



PERFORMANCE CHARECTERISTICS:

Refer to Directional Insert- Meridian Bioscience illumigene HSV 1&2


REFERENCES:

Refer to Directional Insert- Meridian Bioscience illumigene HSV 1&2


Meridian Bioscience

3471 River Hills Drive

Cincinnati, OH 45244 USA



Ph: (800) 343-3858, (513) 271-3700 SN11107 REV 07/15


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