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RealBest-Genetics HLA-B*27


Real time PCR test kit a
to detect the HLA-B?27 allele

USER'S GUIDE

FOR PROFESSIONAL USE
C
D-3836 IVD

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

The kit contains reagents for 48 tests , including control samples .

2. WHO CONTENTS

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

The following types of clinical samples have been validated with the kit: whole blood, buccal swab.

DNA can be extracted from clinical samples using the extraction kits : "RealBest￾Genetics DNA-express", " RealBest GenMag", "RealBest extraction 100" (manufacturer: AO Vector-Best), or other DNA from whole blood and buccal swabs with kits intended for extraction .

PCR master mix (RMM), lyophilized - 48 cs;

The " RealBest -Genetics HLA-B"27" test kit is used to detect the human histocompatibility complex B-locus "27 allele (HLA-B"27) associated with the risk of seronegative spondyloarthritis in clinical samples, real -time polymerase chain reaction (PCR) and using fluorescence detection .

The kit also contains PCR optical quality film - 1 sheet.

1. USE OF ORDINANCE

The method is based on the detection of the HLA-B?27 allele-specific DNA fragment and the DNA fragment corresponding to the autosomal HMBS gene , which is common to all human DNA samples .

PRINCIPLE OF THE METHOD

Control sample (CS) - 1 tsp, 1 ml;

"Genetics 2" sample diluent - 2 vials , 12 ml each ;

Amplification of the HLA-B gene fragment can only be observed if the sample contains the HLA-B"27 allele, regardless of the presence of other alleles of the HLA- B locus .

Amplification of the human HMEBS gene fragment should be observed in each sample , indicating the appropriate human DNA content of the sample and the correctness of the sample preparation procedure .

DNA denaturation , the primer is increasing. PCR consists of repeated cycles : annealing and synthesis of the complementary chain .

4. SPECIFICATIONS

4.1. The DNA markers of the HLA-B"27 allele and the HMBS gene must be detected in the CS .

4.2. The analytical specificity of the "RealBest-Genetics HLA-B"27" test kit is provided by the specific primers and probes . The primers and probes were checked by sequence comparison analysis for possible homologies with all sequences published in genebanks .

4.3. THE

The threshold cycle value (Ct) is the cycle number at which the fluorescence generated in the reaction crosses the threshold value and the fluorescence signal rises significantly above the background . The increased signal is specific for the specific DNA sequence detected by DNA hybridization . thanks to the use of a probe . its fluorescence intensity depends on the initial copy number of the pathogenic DNA template in the sample.

The analytical sensitivity of HLA-B"27 allele detection was determined as follows : the standard reference sample (HLA-B"27- positive) was diluted to obtain samples in which the concentration of human DNA reached the validity threshold (HMBS Ct s 28 ). In this case, all diluted samples were determined as positive (10090) .

44. Diagnostic evaluation

Diagnostic sensitivity of the detection of the HLA-B"27 allele : clinical tests performed on 47 positive samples showed a sensitivity of 10096 ( interval 92.599 - 10096 , with a confidence level of 9590 ) .

5. WARNINGS AND SAFETY PRECAUTIONS

Do not use the kit after the expiration date .

For in vitro use only .

Never use the same pipette tip for different samples.

Do not combine reagents from different batches or from different vials of the same batch .

In order to avoid contamination , the stages of PCR and DNA removal must be spatially separated from each other.

The kit may only be used by qualified personnel .

For reliable results , strictly follow the instructions for use that come with the kit .

When handling the kit , follow the national safety regulations for working with pathogens .

wear when handling kit components is disposable and protective gloves , lab coat and eye protection.

Each workplace must have its own variable volume pipette, the necessary auxiliary materials and equipment. It is forbidden to transfer them to other workplaces .

Use only disposable pipette tips with filters .

Dispose of unused reagents and waste in accordance with national , federal , state , and local regulations .

6. REQUIRED BUT NOT SUPPLIED ADDITIONAL MATERIALS AND EQUIPMENT

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 an extraction kit containing magnetic particles is used, the tubes containing the extracted NA should be held in a magnetic rack .

Each group of samples subjected to the DNA extraction procedure includes a control sample (CS) from this kit .

The CS must follow the same DNA extraction procedure as the control sample (PC) (see the extraction kit instructions for use

7.1. DNA extraction using the extraction kit : "RealBest-Genetics DNA￾express"

Perform DNA extraction according to the extraction kit instructions . For elution , use the "Genetics 2" sample diluent included in the kit . The samples are then ready for PCR .

7.2. DNA extraction with the "RealBest extraction 100" extraction kit

After first opening the vial , the "Genetics 2" sample diluent must be stored at (2-8 ) "C for a maximum of 3 months .

DNA elution should be performed with the 200 ul sample diluent included in the extraction kit . Then add 250 ul of " Genetics 2" Sample Diluent included in the kit . Vortex the tubes thoroughly . Centrifuge at 13,000 rpm for 1 minute . Place the tubes in the magnetic stand.

Then add 400 4I "Genetics 2" Sample Diluent from this kit .

After opening the package for the first time , the RMM must be stored for no more than 3 months .

7.3. DNA extraction using extraction kits from other manufacturers

If you use another kit to extract DNA from whole blood or buccal swabs , perform the procedure according to the kit's instructions for use .

Before the DNA extraction procedure , whole blood samples must be treated with "RealBest Hemolytic " kit . Perform the procedure with 50 ul of whole blood according to the kit's instructions for use .

8. PROCEDURE

8.1. " Preparation of kit components

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

Attention! Put the remaining tubes back immediately, press out the air inside them with the foil bag , and close them tightly with a clamp.

PCR master mix (RMM) sealed in the package (18-26) at "C for at least 30 minutes. Open the package and use scissors to cut the required number of tubes with RMM (counting the test samples and CS ). The tubes cut it together with the cover film .

After first opening the tube , CS 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 .

8.5.

Shake the PCR reaction for 1 minute on a plate shaker at 1200 rpm .

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 :

8.9.

Program the position of the tubes containing test samples and CS according to the instructions for use of the cycler used .

8.10.

Run the program.

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

9. DATA ANALYSIS AND ITS MEANING

9.1.

In the case of CS , the programme:

9.2.

For each test sample , the program should detect the increase in the amplification signal of the HMBS gene ( " FAM" channel) and determine the HMBS Ct value. The sample can be considered valid if Ct in the "FAM" channel is less than or equal to 28 . For invalid samples ( Ct in " FAM " channel above 28 ), repeat the analysis starting from DNA extraction or sampling .

Attention! The analysis results of the test sample can only be considered valid if the sample complies with 9.2 . is valid according to the requirements of point

9.3. Detection of the HLA-B?27 allele

9.3.1.

If an increase in the amplification signal of the HLA-B"27 allele ("ROX" channel) is detected and the HLA-B"27 Ct value is determined for a valid test sample ( according to point 9.2 ), the ACt value is calculated using the following formula :

ACt - HLA-B?"27 Ct - HMBS Ct

9.3.2.

The sample can be considered positive , i.e. it contains the HLA-B"27 allele, if ACt is less than or equal to 7.

9.3.3.

The sample can be considered negative, i.e. it does not contain the HLA -B"27 allele, if the ACt is above 7 , or if the increase in the amplification signal of the HLA-B?27 allele cannot be detected and the HLA-B"27 Ct is not determined .

9.4. Diagnostic value of the test

The HLA-B"27 antigen is of European ethnicity in the genotype of 7-890 representatives and 1-690 of Asians HLA￾B"27 cannot be detected in the majority of HLA-B"27 carriers , however, its frequency is significantly higher in Bechterew's disease (Bechterew's disease), reactive arthritis, psoriatic arthritis, arthritis and (secondary ) uveitis.

The pathogenic role of bacterial pathogens such as Chlamydia trachomatis and gram -negative bacteria (Klebsiella, Salmonella, Yersinia, Shigella and Campylobacter) has been proven by epidemiological and clinical experimental evidence . Microbial antigens can induce autoimmunity through a mechanism of molecular mimicry between the HLA-B"27 antigen and the HLA-B"27 antigen . the lipopolysaccharide of the bacterial cell .

The exact molecular and pathogenetic mechanisms of the relationship between the HLA-B?27 allele and related inflammatory diseases are still not sufficiently clear. Most HLA -B"27 carriers are healthy, although they have a higher risk of rheumatic diseases than HLA - B"27 negative individuals. Manifestations of diseases occur more often in men than in women.

Due to the higher risk of inflammatory joint diseases , carriers of the HLA-B"27 allele must pay particular attention to the prevention and timely treatment of bacterial infections of the intestinal- urogenital tract (especially chlamydiosis ) .

In patients suffering from Reiter's syndrome, enteropathic endogenous uveitis . The HLA-B"27 antigen existence and presence of the allele definitely indicates a formation. A turns away not in a person's genotype exists. The B"27 allele group encodes a version of the antigen that can participate in the autoimmune process against body tissues rich in collagen or proteoglycans . The autoimmune process does not usually start spontaneously , but is initiated by bacterial infection .

fragments of proteins from the immune system must be displayed by the immune system , these are of foreign (bacterial or viral origin ) , the infected cell must be destroyed .

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 TRANSPORT ZS ; GeTOP-6, sZ AA

The kit must be transported on (2-8) "C. At most ; att hmérs KAN

The shelf life of the kit is 18 months from the date of manufacture .

Store the set in the manufacturer's packaging at -(2-8) "C during the entire shelf life .

12. WARRANTY

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

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

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 .

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 .

Authorized representative in Europe:
BIORON GmbH
In den Rauhweiden 20,
67354 Roemerberg, Germany
Tel.: tE-mail: techsupport obioron.net
www.bioron.net

Version: e08.09.23

APPENDIX

Programming the DT-96 and DTprime cyclones :

The metering exposure must be set. Select the Operation with the device mode in the Settings menu , then select the Metering exposure menu up to FAM 250; HEX and ROX up to 1000; Cy5 to 500.

item: yes to Confirm the saving of the current exposure value by pressing the YES button .

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

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