FlexStar® CT & NG PCR Detection Mix 1.5
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Instructions for use FlexStar® CT & NG PCR Detection Mix 1.5 05/2023 EN

FlexStar® CT & NG PCR Detection Mix 1.5 For research use only! (RUO)

1. Application The FlexStar® CT & NG PCR Detection Mix 1.5 is a reagent system, based on realtime PCR technology, for the qualitative detection and differentiation of Chlamydia trachomatis and Neisseria gonorrhoeae specific DNA. For research use only (RUO)! Not for use in diagnostic procedures. 2. Product content The FlexStar® CT & NG PCR Detection Mix 1.5 contains the following components: Table 1: Kit components Nominal volume [μl/tube] Contains biological material of animal origin Positive Control (CT and NG specific DNA) No Template Control (negative control)

3. Storage • The FlexStar® CT & NG PCR Detection Mix 1.5 is shipped on dry ice. The product components should arrive frozen. If one or more components are not frozen upon receipt, or if tubes have been compromised during shipment, contact altona Diagnostics technical support for assistance (see chapter 9. Technical assistance). • All components should be stored at -25 °C to -15 °C upon arrival. • Repeated thawing and freezing of the Detection Mix component should be avoided, as this might affect the performance of the product. • Repeated thawing and freezing of the Positive Control (PC) and...

For CT, a dual-target system, targeting the chromosomal and plasmid DNA, is used. Both targets are detected in the same detection channel. For NG 2 targets are detected in 2 separate detection channels. Probes specific for CT DNA are labeled with the fluorophore FAM™, probes specific for NG Target opa (NG opa) DNA are labeled with the fluorophore ROX™ and probes specific for NG Target porA (NG porA) DNA are labeled with the fluorophore Cy5, respectively. The probe specific for the IC is labeled with the fluorophore JOE™. Using probes linked to distinguishable dyes enables the parallel...

4.2 Real-time PCR instruments The FlexStar® CT & NG PCR Detection Mix 1.5 can be used with the following realtime PCR instruments: • CFX96™ Deep Well Dx System (Bio-Rad) • CFX96™ Dx System (Bio-Rad) • QuantStudio™ 5 Real-Time PCR System (Applied Biosystems) • Rotor-Gene® Q5/6 plex Platform (QIAGEN) NOTE Ensure that all instruments used have been installed, calibrated, checked and maintained according to the manufacturer's instructions and recommendations. 5. Material required but not provided The following additional instruments and consumables are required for use of the FlexStar® CT & NG...

Reagents required but not included in the FlexStar® CT & NG PCR Detection Mix 1.5: • FlexStar® (RT-)PCR Amplification Mix 1.5 (Order No. FS0011503/FS0011505) • AltoStar® Internal Control 1.5 (Order No. IC15-06) 6. Procedure 6.1 Sample preparation Extracted DNA is the starting material for the FlexStar® CT & NG PCR Detection Mix 1.5. The quality of the extracted DNA has a profound impact on the performance of the product. For additional information and technical support regarding pre-treatment and sample preparation, contact altona Diagnostics technical support (see chapter 9. Technical...

► No matter which method/system is used for nucleic acid extraction, never add the IC directly to the sample. The IC should always be added to the sample/ lysis buffer mixture. The volume of the IC which has to be added, always and only depends on the elution volume. It represents 50 % of the elution volume. For instance, if the nucleic acid is going to be eluted in 60 µl of elution buffer or water, 30 µl of IC per sample must be added into the sample/lysis buffer mixture. ► Set up the master mix according to the following pipetting scheme: Table 2: Pipetting scheme (master mix setup)...

Table 3: Pipetting scheme (reaction setup) Reaction setup Master mix Total volume ► Make sure that at least 1 PC and 1 NTC is used per run. ► Thoroughly mix the samples and controls with the master mix by pipetting up and down. ► Close the 96-well reaction plate with appropriate lids or optical adhesive film and the reaction tubes with appropriate lids. ► Centrifuge the 96-well reaction plate in a centrifuge with a microtiter plate rotor and the reaction tubes in an appropriate centrifuge for 30 seconds at approximately 1,000 x g (~ 3,000 rpm). ► The NTC does already contain the IC template...

7.1 Settings ► Define the following settings: Table 4: Run settings Settings Reaction volume Ramp rate Passive reference* 7.2 Fluorescence detectors (dyes) ► Define the fluorescence detectors (dyes): Table 5: Fluorescence detectors Target Detector name Internal Control

7.3 Temperature profile and dye acquisition ► Define the temperature profile and dye acquisition: Table 6: Temperature profile and dye acquisition Stage Cycle repeats Time [min:s] Reverse transcription 8. Data analysis For basic information regarding data analysis on specific real-time PCR instruments, refer to the user manual of the respective instrument. For detailed instructions regarding the analysis of the data generated with the FlexStar® CT & NG PCR Detection Mix 1.5 on different real-time PCR instruments, contact altona Diagnostics technical support (see chapter 9. Technical...

8.1 Interpretation of results 8.1.1 Qualitative analysis Table 7: Result interpretation Detection channel FAM™ (CT) Only CT specific DNA detected. Only NG porA specific DNA detected.2) Neither CT nor NG specific DNA detected. The sample does not contain detectable amounts of CT or NG specific DNA. PCR inhibition or reagent failure. Repeat testing from original sample or collect and test a new sample. Result interpretation * Detection of the IC in the JOE™ detection channel is not required for positive results in the FAM™ and/or the ROX™ and/or the Cy5 detection channel. A high CT and/or NG...

Data analysis using the Rotor-Gene® Q5/6 plex Platform (QIAGEN): Using the Rotor-Gene® Q5/6 plex Platform (QIAGEN) a slight crosstalk between the orange (ROX™) detection channel and the red (Cy5) detection channel might be observed. This can lead to the following signal phenotype. CT DNA specific signals will show up only in the green (FAM™) detection channel, NG porA DNA specific signals will show up only in the red (Cy5) detection channel, internal control specific signals will show up only in the yellow (JOE™) detection channel. In contrast, NG opa DNA specific signals might not only...