RealBest DNA Staphylococcus aureus/mecA/lukS-PV

Real-time PCR test kit for Staphylococcus aureus DNA, methicillin resistance (mecA) and Panton-Valentin leukocidin (lukS-PV) to detect coding genes

FOR PROFESSIONAL USE
D-5603 I bet
USER'S GUIDE

1. USE OF ORDINANCE

The "RealBest DNA Staphylococcus aureus/mecA/lukS-PV" test kit is used for the detection of Staphylococcus aureus DNA and genes encoding methicillin resistance (mecA) and Panton-Valentin leukocidin (lukS-PV) in clinical samples, surface swabs, in bacterial and blood cultures using real-time polymerase chain reaction (PCR) and fluorescence detection.

PRINCIPLE OF THE METHOD

Real-time PCR is based on the detection of fluorescence produced by the reporter molecule, which increases as the reaction progresses. The report molecule is a two-tag is a DNA probe that specifically binds to the target region of the pathogen's DNA. The fluorescence signal increases due to the separation of fluorescent dye and quencher during amplification due to the exonuclease activity of Tag DNA polymerase. PCR consists of repeated cycles; DNA denaturation, primer annealing and complementary strand synthesis.

2. CONTENTS OF KIT

The kit contains reagents for 96 tests, including control samples.

PCR master mix (RMM), lyophilized - 96 cs;
Negative control sample (NC) - 2 vials, 1 ml each;
Positive control sample (PC) - 1 tsp, 1 ml;

The kit also includes PCR optical quality film - 1.5 sheets.

The kit is intended for use with block-type PCR cyclers: CFX96 (Bio-Rad, USA), DT-96, DTprime and DTIite (DNA-Technology, Russia).

The following types of samples were validated with the kit: blood plasma, mucosa swabs, urine, sputum, nasopharyngeal aspirate, bronchoalveolar lavage fluid, wound drainage, cerebrospinal fluid, biopsy material, surface swabs, bacterial and blood cultures.

DNA can be extracted from the samples using the extraction kits: "RealBest DNA-express", "RealBest extraction 100" or "RealBest UniMag" (manufacturer: AO Vector-Best). When using NA extraction kits from other manufacturers, it is strongly recommended to use the internal control sample (IC, REF C-8881, AO Vector-Best), which you can order additionally. Please note that without IC it is not possible to interpret a complete and valid result.

The internal control sample (IC) is used to control the possibility of false negative results (due to DNA loss during the extraction procedure or the inhibitory effect of sample components). IC should be added to each sample and control sample prior to the DNA extraction procedure.

The results of the PCR analysis are taken into account in the complex diagnosis of diseases.

3. SPECIFICATIONS

4.1. The analytical specificity of the "RealBest DNA Staphylococcus aureus/mecA/lukS-PV" assay kit is ensured by specific primers and probes.

Primers and probes were checked for possible homologies with all sequences published in genebanks by sequence comparison analysis. In addition, cross-reactivity was assessed using a control panel containing DNA from closely related species. No false positive results were obtained.

4.2. The analytical sensitivity of the detection of Staphylococcus aureus DNA, mecA and lukS-PV genes was determined using probit analysis and verified on the following types of samples:

blood plasma, mucosal swab, urine, sputum, nasopharyngeal aspirate, bronchoalveolar lavage fluid, wound drainage, liguor, biopsy material, surface swab, bacterial and blood culture.

Note: Analytical sensitivity depends on sample volume, elution volume, NA extraction method and other factors.

4.3. Diagnostic evaluation

Diagnostic sensitivity of Staphylococcus aureus DNA detection: the clinical test performed on 37 positive samples (blood plasma, mucosal swab, urine, sputum, nasopharyngeal and laryngeal aspirate, bronchoalveolar lavage fluid, wound drainage, ligure, biopsy material, surface swab, bacterial and blood culture) showed a sensitivity of 100% (interval 90.5% - 100%, with a confidence level of 95%).

Diagnostic specificity of Staphylococcus aureus DNA detection: clinical tests performed on 53 negative samples (blood plasma, mucosal swab, urine, sputum, bronchoalveolar lavage fluid, wound drainage, liguor, biopsy material, surface swab, bacterial and blood culture) showed a specificity of 100% (Interval 93.3% - 100%, with a confidence level of 95%).

Diagnostic sensitivity of mecA gene detection: clinical tests performed on urine, sputum, nasopharyngeal and nasal mucosa (blood plasma, aspirate, bronchoalveolar lavage fluid, wound drainage, cerebrospinal fluid, biopsy material, surface swab, bacterial and blood cultures) showed a sensitivity of 100% (interval 95% - 100%, with a confidence level of 95%).

Diagnostic specificity of mecA gene detection: on the 56 negative samples (blood plasma, mucous membrane swab, urine, sputum, nasopharyngeal and nasal mucosa aspirate, bronchoalveolar lavage fluid, wound drainage, cerebrospinal fluid, biopsy material, surface swab, bacterial and blood culture) showed a specificity of 100% (interval 93.6% - 100%, with a confidence level of 95%).

Diagnostic sensitivity of lukS-PV gene detection: clinical tests performed on 10 positive samples (blood plasma, mucous membrane swab, urine, sputum, nasopharyngeal aspirate, bronchoalveolar lavage fluid, wound drainage) showed a sensitivity of 100% (interval 69.2% - 100%, with a confidence level of 95%).

The diagnostic specificity of lukS-PV gene detection: on the 80 negative samples (blood plasma, mucous membrane swab, urine, sputum and nasal mucosa aspirate, bronchoalveolar lavage fluid, wound drainage, cerebrospinal fluid, biopsy material, surface swab, bacterial and blood culture) showed a specificity of 100% (interval 95.5% - 100%, with a confidence level of 95%).

The presence of the lukS-PV gene in the samples found positive by the test kit was confirmed by Sanger sequencing.

Analysis with a CE-marked reference kit showed complete agreement.

5. WARNINGS AND SAFETY PRECAUTIONS

For in vitro use only.
Follow national pathogen safety guidelines when handling the kit.
The set may only be used by qualified personnel.
Do not use the kit after the expiry date.
Do not combine reagents from different batches or from different vials of the same batch.
Wear disposable gloves, a lab coat, and eye protection when handling samples and kit components.
Use only disposable pipette tips with filters.
Never use the same pipette tip for different samples.
Each workplace must have its own variable volume pipette, the necessary auxiliary materials and equipment. It is forbidden to transfer them to other workplaces.
In order to prevent contamination, the PCR and DNA removal stages must be spatially separated from each other.
Do not use the Kits from different lots component in an experiment.
Dispose of unused reagents and waste in accordance with national, federal, state, and local regulations.
For reliable results, strictly follow the instructions for use that come with the kit.

6. REQUIRED BUT NOT SUPPLIED ADDITIONAL MATERIALS AND EQUIPMENT

DNA extraction kit;
Internal control sample (AO Vector-Best, REF C-8881) when using another manufacturer's NA extraction kit;
Real-time PCR cycler;
Semi-automatic variable volume pipettes and disposable tips;
Microcentrifuge;
Vortex mixer;
Laminar safety box;
Racks for 2 ml and 0.2 ml tubes;
Disc mixer, which provides a rotation speed of 1200 revolutions per minute;
Refrigerator;
Disposable medical, non-sterile, dust-free gloves;
Biohazardous waste container;
Scissors.

7. SAMPLE PREPARATION

The test must be performed on DNA samples extracted from clinical material using one of the DNA extraction kits listed on page 1, in accordance with the kit's instructions for use. If using an extraction kit containing magnetic particles, keep the tubes with the extracted NA in a magnetic rack.

Each group of samples subjected to the DNA extraction procedure includes a positive control sample (PC) and a negative control sample (NC) from this set.

The extracted DNA is stored at (2-8) °C for a maximum of 24 hours.

After first opening the vial, NC should be stored at (2-8) °C for a maximum of 3 months.

After first opening the tube, PC must be stored at (2-8) °C for a maximum of 3 months.

8. PROCEEDINGS

Before analysis, remove the kit from the refrigerator and keep ready.

8.1. Preparing the components of the set

PCR Master Mix (RMM) sealed in the package (18-26) °C for at least 30 minutes. Open the package and use scissors to cut the required number of tubes with RMM (counting test samples and control samples: 1 NC and 1 PC) Cut the tubes together with the cover film.

Attention! Immediately put the remaining tubes back into the foil bag, squeeze out the air, and seal tightly with a pinch.

After opening the package for the first time, RMM must be stored at (2-8) °C for a maximum of 3 months.

8.2. Label the tubes with RMM for each test and control sample.

Attention! Labels should be placed on the side of the pipes.

8.3. Carefully remove the cover sheet leaving the RMM in the tube.

8.4. Add 50 µl of the appropriate extracted DNA solution to each tube using a new pipette tip fitted with a filter. Seal the tubes tightly with PCR optical grade foil. Start the PCR no later than 30 minutes after adding the DNA.

Attention! The optical film must remain clean (free from labels and marks).

8.5. Shake the PCR plate for 1 minute on the plate shaker at 1200 rpm speed.

8.6. Place the tubes in the real-time PCR cycler.

8.7. Program the real-time PCR cycler as follows:

Stage 1: 50 °C - 2 minutes;
Stage 2: 95 °C - 2 minutes;
Stage 3: 50 cycles (94 °C - 10 sec; 60 °C - 20 sec).

Measurement of fluorescence at 60 °C.

8.8. Select the detection channels:

detection of the mecA gene amplification signal in the "FAM" channel;
an increase in the amplification signal of the mecA gene ("FAM" channel) and a
determination of PC Ct;
the amplification signal of Staphylococcus aureus DNA in the "ROX" channel;
an increase in the DNA amplification signal of Staphylococcus aureus
detection of the lukS-PV gene amplification signal in the "HEX" channel;
increase of lukS-PV gene amplification signal ("HEX" channel) and determination of PC Ct;
detect IC DNA amplification signal in "Cy5" channel;
detection of IC DNA amplification signal increase ("Cy5" channel) and determination of IC Ct.

8.9. Run the program.

8.10.

For PC, the program:

detects and determines the PC Ct value;
detects the increase in IC DNA amplification signal ("Cy5" channel)and determination of IC Ct.

For NC, the program should detect the increase in IC DNA amplification signal ("Cy5" channel) and determine the IC Ct value. Neither "ROX", "FAM", nor "HEX" fluorescence signal should appear up to 36 Ct. If Ct on "ROX" or "FAM" or "HEX" channel is less than or equal to 36, see 9.10. point.

For each test sample, the program should detect the increase in IC DNA amplification signal ("Cy5" channel) and determine the IC Ct value.

9. DATA ANALYSIS AND INTERPRETATION

9.1.

Calculate (IC Ct)_av as the average IC Ct value of all samples (including PC and NC). Those IC Ct values that are more than

They differ from the (IC Ct)_av value by 2 and should be ignored. Recalculate (IC Ct)_av for the remaining values.

9.2. The sample can be considered positive, i.e. as containing Staphylococcus aureus DNA with a channel Ct value of "ROX" less than or equal to 32, if the additional criteria are met (see Table 1).

9.3. The sample can be considered positive, i.e. it contains the mecA gene, if the Ct value of "FAM" is less than or equal to 32.

9.4. The sample can be considered positive, i.e. it contains the lukS-PV gene, if the Ct value of "HEX" is less than or equal to 32.

9.5. If the "ROX" or "FAM" or "HEX" channel Ct value is above 32, but maximum 36, then the additional criteria must be applied (see Table 1).

Ct value of the test sample	(NC Ct) or (NC Ct-X Ct)	Interpretation
X Ct ≤ 32	-	positive
32 < X Ct ≤ 36	≤ 24	positive
32 < X Ct ≤ 36	> 24	undetermined
NC Ct ≤ 36	-	negative

9.6. If the sample cannot be considered unambiguous, i.e. it contains target DNA in a concentration of less than 103 CFU/ml, a repeated analysis of the sample is required, starting with the DNA extraction step. If the uneven result is still

9.7. Additional criteria

9.8. To interpret results

The sample can be viewed as negative, i.e. as not containing Staphylococcus aureus DNA if the Ct value of "ROX", "FAM" and "HEX" channel is undefined or (X Ct) is above 36.

9.9. Ambiguous results

If the NC Ct value is less than or equal to 36 in the "ROX" or "FAM" or "HEX" channel, it indicates the presence of contamination. In this case, all positive results of a given individual PCR test are considered doubtful results. Measures must be taken to identify and eliminate the source of contamination.

Repeat the test on all positive samples from that run.

Samples that showed a negative result in this run should be considered negative.

9.10.

If the NC Ct value is less than or equal to 36 in the "ROX" or "FAM" or "HEX" channel, it indicates the presence of contamination.

9.11. Diagnostic value of mecA and lukS-PV genes detection

Methicillin-resistant strains of Staphylococcus aureus are resistant to several drugs and cause difficult-to-treat conditions such as sepsis and pneumonia.

Strains of Staphylococcus aureus that produce the pore-forming toxin Panton-Valentine leukocidin (PVL) cause neucrotizing pneumonia, a rare but very serious condition with high mortality.

10. QUALITY CONTROL

AO Vector-Best's ISO 13485 certified quality management system requires quality control to be performed on each batch produced to ensure consistent product quality.

11. STORAGE AND DELIVERY

Store the set in the manufacturer's packaging (2-8) °C during the entire shelf life. The shelf life of the set is 18 months from the date of manufacture.

The stock must be delivered at a temperature of (2-8) °C for a maximum of 10 days. Take up to 26 °C.

12. WARRANTY

The manufacturer hereby guarantees that the manufactured products meet the requirements of the normative and technical documentation.

The safety and quality of the products are guaranteed during the entire shelf life.

The manufacturer is responsible for the non-compliant properties of the product, with the exception of those defects that arose as a result of violation of the instructions for use, transport and storage conditions, the activities of third parties or force majeure.

The manufacturer is obliged to replace the product at his own expense if the technical and functional characteristics of the product do not comply with the normative and technical documentation, and these disadvantages are caused by a hidden material defect or manufacturing defect.

APPENDIX

Programming the DT-96, DTprime and DTIite cyclers:

The metering exposure must be set. Select the Operation with the device mode in the Settings menu, then select the Metering exposure menu item:

FAM up to 250; HEX and ROX up to 1000; Cy5 to 500.

Confirm saving the current exposure value by pressing the YES button.

Attention! The exposure values given are for RealBest kits only and should be modified for other purposes if necessary.

Authorized representative in Europe:
BIORON GmbH
In den Rauhweiden 20,
67354 Roemerberg, Germany
Tel.: +49 62 37 92 74 40
E-mail: techsupport@bioron.net
www.bioron.net

Version: 08.09.23

RealBest DNA Staphylococcus aureus/mecA/lukS-PV