BD ProbeTec Controls

INTENDED USE

The BD ProbeTec ET Neisseria gonorrhoeae (GC) Amplified DNA Assay when tested with the BD Viper™ System, uses Strand Displacement Amplification (SDA) technology for the direct, qualitative detection of Neisseria gonorrhea DNA in endocervical swabs, male urethral swabs, and in female and male urine specimens as evidence of infection with N. gonorrhoeae. Specimens may be from symptomatic or asymptomatic females and males. A separate Amplification Control is used for inhibition testing.

SUMMARY AND EXPLANATION

Neisseria gonorrhoeae are gram-negative, oxidase-positive diplococci which can be observed in Gram-stained smears of urethral discharges, usually within neutrophils. The culture of N. gonorrhoeae can be difficult because the organism does not survive long outside its host and is highly susceptible to adverse environmental conditions such as drying and extreme temperatures.1 Neisseria gonorrhoeae causes acute urethritis in males, which if untreated can develop into epididymitis, prostatitis, and urethral stricture. In females, the primary site of infection is the endocervix. An important complication in females is the development of pelvic inflammatory disease which contributes to infertility.2 Asymptomatic infections occur often in females but infrequently in males.

The current methods for the detection of N. gonorrhoeae include culture, immunoassays, non-amplified probes, and amplified probes.1,2 The development of amplified methods has demonstrated two advantages over non-amplified methods: increased sensitivity, and applicability to a variety of sample types. For the identification of GC, optimized culture methods continue to be the standard for diagnosing patients with gonococcal infections. The BD ProbeTec ET Neisseria gonorrhoeae Amplified DNA Assay, when used with the BD Viper System, utilizes homogeneous Strand Displacement Amplification (SDA) technology as the amplification method and fluorescent energy transfer (ET) as the detection method to test for the presence of N. gonorrhoeae DNA in clinical specimens.3-5

PRINCIPLES OF THE PROCEDURE

The BD ProbeTec ET Neisseria gonorrhoeae Amplified DNA Assay is based on the simultaneous amplification and detection of target DNA using amplification primers and a fluorescent-labeled detector probe.4,5 The SDA reagents are dried in two separate disposable microwell strips. The processed sample is added to the Priming Microwell which contains the amplification primers, fluorescent-labeled detector probe, and other reagents necessary for amplification. After incubation, the reaction mixture is transferred to the Amplification Microwell, which contains two enzymes (a DNA polymerase and a restriction endonuclease) necessary for SDA.

The Amplification Microwells are sealed to prevent contamination and then incubated in a thermally controlled fluorescent reader which monitors each reaction for the generation of amplified products. The presence or absence of GC is determined by relating the BD ProbeTec ET MOTA (Method Other Than Acceleration) scores for the sample to pre-determined cutoff values. The MOTA score is a metric used to assess the magnitude of the signal generated as a result of the reaction. Each sample and control are tested in two discrete microwells: one for N. gonorrhea and one for the Amplification Control. The purpose of the Amplification Control is to identify a sample that may inhibit the SDA reaction.

REAGENTS

Each BD ProbeTec ET GC/AC Reagent Pack contains:

Neisseria gonorrhoeae (GC) Priming Microwells, 4 x 96:

4 Oligonucleotides ≥ 7 pmol; dNTP ≥ 35 nmol; Detector probe ≥ 25 pmol; with buffers and stabilizers.

Neisseria gonorrhoeae (GC) Amplification Microwells, 4 x 96:

Restriction enzyme ≥ 15 Units; DNA Polymerase ≥ 2 Units; dNTP’s ≥ 80 nmol; with buffers and stabilizers.

Amplification Control (AC) Priming Microwells, 4 x 96:

4 Oligonucleotides ≥ 7 pmol; dNTP ≥ 35 nmol; Detector probe ≥ 25 pmol; ≥ 1,000 copies per reaction of pGC10

linearized plasmid; with buffers and stabilizers.

Amplification Control (AC) Amplification Microwells, 4 x 96:

Restriction enzyme ≥ 15 Units; DNA Polymerase ≥ 2 Units; dNTP’s ≥ 80 nmol; with buffers and stabilizers.

NOTE: Each microwell pouch contains one desiccant bag.

BD ProbeTec ET (CT/GC) Control Set, 20 CT/GC Positive Controls (50 µL dried) containing 750 copies per reaction of
pCT16 linearized plasmid* and 250 copies per reaction of pGC10 linearized plasmid* with ≥ 5 µg Salmon testes DNA;
20 CT/GC Negative Controls (50 µL dried) with ≥ 5 µg Salmon testes DNA; BD ProbeTec ET CT/GC Diluent Tubes – 400
tubes each containing 2 mL of Sample Diluent, which contains potassium phosphate, DMSO, glycerol, Polysorbate 20,

and 0.03% Proclin™ (preservative); BD ProbeTec ET Diluent (CT/GC) – 225 mL Sample Diluent which contains
potassium phosphate, DMSO, glycerol, Polysorbate 20, and 0.03% Proclin (preservative). * The concentration of this DNA was determined spectrophotometrically at 260 nm. The instrument, equipment, and supplies: BD Viper, BD Viper Lysing Heater, BD Viper Lysing Rack and base, BD ProbeTec Urine Preservative Transport Kit, BD ProbeTec ET Sample Tubes and Caps.

BD Viper pipette tips, tip waste boxes and bags, Amplification (Black) plate sealers, BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhoeae (CT/GC) Amplified DNA Assay Endocervical Specimen Collection and DRY TRANSPORT Kit or BD ProbeTec ET CT/GC Amplified DNA Assay Collection Kit for Endocervical Specimens, BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhoeae CT/GC Amplified DNA Assay Male Urethral Specimen Collection and DRY TRANSPORT Kit or BD ProbeTec ET CT/GC Amplified DNA Assay Collection Kit for Male Urethral Specimens.

Materials Required But Not Provided: Centrifuge capable of 2000 x g, vortex mixer, gloves, pipettes capable of delivering 1 mL, 2 mL and 4 mL, DNA AWAY™ or 1% (v/v) sodium hypochlorite, clean container suitable for holding aliquotted Diluent, timer and absorbent paper, sterile urine specimen collection cups. Empty microwells and alcohol wipes (70% Isopropanol). *Mix 200 mL of bleach with 800 mL of water. Storage and Handling Requirements: Reagents may be stored at 2 – 33°C. Unopened Reagent Packs are stable until the expiration date. Once a pouch is opened, the microwells are stable for 4 weeks if properly sealed or until the expiration date, whichever comes first. Do not freeze.

Warnings and Precautions:

  1. For in vitro Diagnostic Use
  2. Wear personal protective equipment, including eye protection, when handling biological specimens.
  3. This reagent pack is for testing endocervical and male urethral swabs and male and female urine specimens with
    the BD Viper System.
  4. For a collection of endocervical swab specimens, only the BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhea (CT/GC) Amplified DNA Assay Endocervical Specimen Collection and DRY TRANSPORT Kit and the BD ProbeTec ET CT/GC Amplified DNA Assay Collection Kit for Endocervical Specimens have been validated.
  5. For a collection of male urethral swab specimens, only the BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhea (CT/GC) Amplified DNA Assay Male Urethral Specimen Collection and DRY TRANSPORT Kit and the BD ProbeTec ET CT/GC Amplified Assay Collection Kit for Male Urethral Specimens have been validated.
  6. For urine specimens, the BD ProbeTec Urine Preservative Transport (UPT), and unpreserved (neat) urine have been validated.
  7. The BD ProbeTec Urine Preservative Transport (UPT) Kit contains NAP Guard™ (≥ 742.5 mM K2EDTA). NAP Guard may be irritating to the eyes, skin, and respiratory system. In case of contact with eyes, rinse opened eye immediately with plenty of water and seek medical advice if symptoms persist. After contact with skin, wash immediately with plenty of soap and water. If inhaled, seek medical attention in case of problems.
  8. Laboratories may validate other swab or urine collection and transport devices for use with the BD ProbeTec ET GC assay according to the “Verification and Validation Procedures in the Clinical Microbiology Laboratory,” Cumitech 31, B.L. Elder et al., American Society for Microbiology, Washington D.C., February 1997.
  9. Do not test the CT/GC Diluent tube from the BD ProbeTec ET CT/GC Amplified Assay Collection Kits if received in the laboratory without the swab present. A false-negative test result may occur.
  10. Do not interchange or mix kit reagents from kits with different lot numbers.
  11. Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical specimens. “Standard Precautions” 6-9 and institutional guidelines should be followed in handling all items contaminated with blood and other body fluids.
  12. Use established laboratory practices when disposing of used pipette tips, sample tubes, Priming Microwells, and other disposables. Discard disposables carefully. Seal and dispose of waste containers when they are 3/4 full or daily (whichever comes first).
  13. The BD ProbeTec ET Diluent (CT/GC) and CT/GC Diluent tube contain dimethyl sulfoxide (DMSO). DMSO is harmful by inhalation, contact with skin or if swallowed. Avoid contact with eyes. In case of contact with the eyes, rinse immediately with plenty of water and seek medical advice. After contact with skin, wash immediately with plenty of water.
  14. Reagent pouches containing unused Priming Microwells and Amplification Microwells MUST be carefully resealed after opening. Verify that a desiccant is present prior to resealing the reagent pouches.
  15. The plate containing the Amplification Microwells MUST be properly sealed with the black Amplification Sealer. Sealing ensures a closed reaction for application and detection and is necessary to avoid contamination of the instrument and work area with amplification products. Do not remove sealing material from microwells at any time.
  16. Priming Microwells with residual fluid (after transfer of fluid from the Priming Microwells to the Amplification Microwells) represents a source of target contamination. Carefully seal Priming Microwells with a plate sealer prior to disposal.
  17. To prevent contamination of the work environment with amplification products, use the disposal bags provided in the Reagent Packs to dispose of tested Amplification Microwells. Make sure the bags are properly closed before disposal.
  18. Although dedicated work areas are not required because the BD Viper System design reduces the possibility of amplicon contamination in the testing environment, other precautions for controlling contamination, particularly to avoid contamination of specimens during processing, are necessary.
  19. Because of the potential for false positivity with some non-gonococcal Neisseria found in the respiratory tract (see “Limitations of the Procedure,” #19), contamination of reagents and specimens with respiratory aerosols should be avoided.
  20. CHANGE GLOVES after removing and discarding caps from lysed samples and controls to avoid cross-contamination of specimens. If gloves come in contact with the specimen or appear to be wet, immediately change gloves to avoid contaminating other specimens. Change gloves before leaving the work area and upon entry into the work area.
  21. In the event of contamination of the work area or equipment with samples or controls, thoroughly clean the contaminated area with DNA AWAY or 1% (v/v) sodium hypochlorite and rinse thoroughly with water. Allow surface to dry completely before proceeding.
  22. Clean the entire work area – countertops and instrument surfaces – with DNA AWAY or 1% (v/v) sodium hypochlorite on a daily basis. Thoroughly rinse with water. Allow surfaces to dry completely before proceeding with additional testing.
  23. Do not use ELIMINase or Alconox for cleaning the BD Viper System.
  24. When using hydrogen peroxide as a cleaning agent, do not use hydrogen peroxide from a bottle that has been open > 8 days.
  25. Contact Technical Services in the event of an unusual situation, such as a spill into the BD Viper instrument or DNA contamination that cannot be removed by cleaning.and 0.03% Proclin™ (preservative); BD ProbeTec ET Diluent (CT/GC) – 225 mL Sample Diluent which contains potassium phosphate, DMSO, glycerol, Polysorbate 20, and 0.03% Proclin (preservative). * The concentration of this DNA was determined spectrophotometrically at 260 nm.
  26. Instrument, equipment and supplies: BD Viper, BD Viper Lysing Heater, BD Viper Lysing Rack and base, BD ProbeTec Urine Preservative Transport Kit, BD ProbeTec ET Sample Tubes and Caps. BD Viper pipette tips, tip waste boxes and bags, Amplification (Black) plate sealers, BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhoeae (CT/GC) Amplified DNA Assay Endocervical Specimen Collection and DRY TRANSPORT Kit or BD ProbeTec ET CT/GC Amplified DNA Assay Collection Kit for Endocervical Specimens, BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhoeae CT/GC Amplified DNA Assay Male Urethral Specimen Collection and DRY TRANSPORT Kit or BD ProbeTec ET CT/GC Amplified DNA Assay Collection Kit for Male Urethral Specimens.
  27. Materials Required But Not Provided: Centrifuge capable of 2000 x g, vortex mixer, gloves, pipettes capable of delivering 1 mL, 2 mL and 4 mL, DNA AWAY™ or 1% (v/v) sodium hypochlorite, clean container suitable for holding aliquotted Diluent, timer and absorbent paper, sterile urine specimen collection cups. Empty microwells and alcohol wipes (70% Isopropanol). *Mix 200 mL of bleach with 800 mL of water. Storage and Handling Requirements: Reagents may be stored at 2 – 33°C. Unopened Reagent Packs are stable until the expiration date. Once a pouch is opened, the microwells are stable for 4 weeks if properly sealed or until the expiration date, whichever comes first. Do not freeze. Warnings and Precautions: For in vitro Diagnostic Use

Wear personal protective equipment, including eye protection, when handling biological specimens.

This reagent pack is for testing endocervical and male urethral swabs and male and female urine specimens with
the BD Viper System.

For a collection of endocervical swab specimens, only the BD ProbeTec ET Chlamydia trachomatis/Neisseria
gonorrhea (CT/GC) Amplified DNA Assay Endocervical Specimen Collection and DRY TRANSPORT Kit and the
BD ProbeTec ET CT/GC Amplified DNA Assay Collection Kit for Endocervical Specimens have been validated.

For a collection of male urethral swab specimens, only the BD ProbeTec ET Chlamydia trachomatis/Neisseria gonorrhea (CT/GC) Amplified DNA Assay Male Urethral Specimen Collection and DRY TRANSPORT Kit and the BD ProbeTec ET CT/GC Amplified Assay Collection Kit for Male Urethral Specimens have been validated.

For urine specimens, the BD ProbeTec Urine Preservative Transport (UPT), and unpreserved (neat) urine have been validated.

The BD ProbeTec Urine Preservative Transport (UPT) Kit contains NAP Guard™ (≥ 742.5 mM K2EDTA). NAP Guard may be irritating to the eyes, skin, and respiratory system. In case of contact with eyes, rinse opened eye immediately with plenty of water and seek medical advice if symptoms persist. After contact with skin, wash immediately with plenty of soap and water. If inhaled, seek medical attention in case of problems.

Laboratories may validate other swab or urine collection and transport devices for use with the BD ProbeTec ET GC assay according to the “Verification and Validation Procedures in the Clinical Microbiology Laboratory,” Cumitech 31, B.L. Elder et al., American Society for Microbiology, Washington D.C., February 1997.

Do not test the CT/GC Diluent tube from the BD ProbeTec ET CT/GC Amplified Assay Collection Kits if received in
the laboratory without the swab present. A false-negative test result may occur.

Do not interchange or mix kit reagents from kits with different lot numbers.

Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical specimens. “Standard Precautions” 6-9 and institutional guidelines should be followed in handling all items contaminated with blood and other body fluids.

Use established laboratory practices when disposing of used pipette tips, sample tubes, Priming Microwells, and other disposables. Discard disposables carefully. Seal and dispose of waste containers when they are 3/4 full or daily (whichever comes first). 12. The BD ProbeTec ET Diluent (CT/GC) and CT/GC Diluent tube contain dimethyl sulfoxide (DMSO). DMSO is harmful by inhalation, contact with skin or if swallowed. Avoid contact with eyes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. After contact with skin, wash immediately with plenty of water.

Reagent pouches containing unused Priming Microwells and Amplification Microwells MUST be carefully resealed after opening. Verify that a desiccant is present prior to resealing the reagent pouches.

The plate containing the Amplification Microwells MUST be properly sealed with the black Amplification Sealer. Sealing ensures a closed reaction for application and detection and is necessary to avoid contamination of the instrument and work area with amplification products. Do not remove sealing material from microwells at any time.

Priming Microwells with residual fluid (after transfer of fluid from the Priming Microwells to the Amplification Microwells) represent a source of target contamination. Carefully seal Priming Microwells with a plate sealer prior to disposal.

To prevent contamination of the work environment with amplification products, use the disposal bags provided in the Reagent Packs to dispose of tested Amplification Microwells. Make sure the bags are properly closed before disposal.

Although dedicated work areas are not required because the BD Viper System design reduces the possibility of amplicon contamination in the testing environment, other precautions for controlling contamination, particularly to avoid contamination of specimens during processing, are necessary.

Because of the potential for false positivity with some non-gonococcal Neisseria found in the respiratory tract (see “Limitations of the Procedure,” #19), contamination of reagents and specimens with respiratory aerosols should be avoided.
19. CHANGE GLOVES after removing and discarding caps from lysed samples and controls to avoid cross-contamination of specimens. If gloves come in contact with the specimen or appear to be wet, immediately change gloves to avoid contaminating other specimens. Change gloves before leaving a work area and upon entry into
the work area.

In the event of contamination of the work area or equipment with samples or controls, thoroughly clean the contaminated area with DNA AWAY or 1% (v/v) sodium hypochlorite and rinse thoroughly with water. Allow surface to dry completely before proceeding.

Clean the entire work area – countertops and instrument surfaces – with DNA AWAY or 1% (v/v) sodium hypochlorite on a daily basis. Thoroughly rinse with water. Allow surfaces to dry completely before proceeding with additional testing.

Do not use ELIMINase or Alconox for cleaning the BD Viper System.

When using hydrogen peroxide as a cleaning agent, do not use hydrogen peroxide from a bottle that has been open > 8 days.

Contact Technical Services in the event of an unusual situation, such as a spill into the BD Viper instrument or DNA contamination that cannot be removed by cleaning.

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