HLA-B27 PCR Assay

The HLA-B27 RealFast Assay is a non-automated real-time PCR test for the detection of HLA-B*27 alleles, specific variants of the HLA-B gene. The qualitative assay discriminates the presence or absence of HLA-B*27 in a human genomic DNA extract from blood and detects the majority of disease-relevant HLA-B*27 subtypes.

What the assay detects, and what it does not

The IFU lists the HLA-B*27 alleles covered, from B*27:01 through B*27:256, and names per group the alleles for which detection is abrogated or reduced, among them B*27:05:09, B*27:07:06, B*27:16, B*27:18, B*27:23 and B*27:26. Other genetic variants in a sample cannot be detected, and a rare or private polymorphism under a primer or probe binding site can cost amplification or fluorescence. For fifteen named HLA-B alleles, including B*27:03 and B*27:17, the HEX control probe may fail in homozygous samples. In silico analysis flags B*07:428, B*14:57, B*15:594, B*37:60, B*44:97 and B*55:121 as likely to cross-react, and for those the manufacturer states that experimental confirmation is still pending. Reference sequence NG_023187.1.

Method

The assay is a 5′ nuclease (TaqMan) test. Gene-specific primers amplify a 202 bp fragment of HLA-B, and two dual-labelled hydrolysis probes report on it. A sample positive for HLA-B*27 gives a signal in both the FAM channel (520 nm) and the HEX channel (556 nm); a negative sample gives a HEX signal only, with no or baseline signal in FAM. The master mix is 10 µl RealFast 2x Genotyping Mix plus 5 µl HLA-B27 Assay Mix, and 5 µl of DNA or control template completes a 20 µl reaction. Set the FAM threshold just above the background of the supplied Negative Control; a sample crossing the threshold beyond Cq 37 is invalid and must be repeated. A no-template control is run in every experiment, alongside the supplied positive and negative controls.

Sample and DNA

Fresh or frozen blood with EDTA anticoagulant is used. Blood with heparin or citrate has not been tested. Blood is not stored more than three days at ambient temperature or more than one week at 2 to 8 °C, and blood frozen for more than a year or thawed more than three times is not used. DNA extraction reagents are not supplied; ViennaLab recommends the GENXTRACT Blood DNA Extraction System (REF 2-014) or the Spin Micro DNA Extraction Kit (REF 2-020). For accurate genotype calling the DNA should be 2 to 10 ng/µl with an OD260/280 ratio of 1.7 to 2.0; higher concentrations are diluted before use.

Run and instruments

Three minutes at 95 °C, then 40 cycles of 15 seconds at 95 °C and one minute at 60 °C, with data acquisition on FAM and HEX. The assay is validated on the AB 7500 Fast, StepOne, CFX96, LightCycler 480, Mx3005P, MIC qPCR Cycler and Rotor-Gene 6000. A 36-well Rotor-Gene rotor needs an adapted programme with annealing at 56 °C, and instruments that normalise on ROX, such as the StepOne, 7300 and 7900, need ROX added to the Genotyping Mix at 1 µM. Because heating and cooling rates differ between instruments, Cq values differ as well and other cyclers have to be verified by the user.

Performance from the IFU

Accuracy was determined on 198 pretyped genomic DNA samples against sequence-specific primer PCR and a commercial HLA-B*27 real-time PCR kit. The assay called all 66 positives and all 132 negatives correctly, so 100 per cent positive and 100 per cent negative percent agreement. Precision covered 296 tests across replicates, operators, days, thermocyclers, reagent lots and extraction methods, all with matching genotypes. The limit of detection is 0.2 ng DNA per reaction. Haemoglobin below 0.2 µM, immunoglobulin G at 0.1 µM, peripheral blood below 0.02 per cent, ethanol at 2 per cent and EDTA at 0.1 mM did not interfere. In the clinical evaluation, a cross-sectional study of 83 ankylosing spondylitis patients, HLA-B*27 was detected in 66.3 per cent.

Storage and use

The kit ships on cooling blocks and is stored at -30 to -15 °C, or at 2 to 8 °C for up to one month, and withstands up to 20 freeze-thaw cycles. Avoid exposure to intense light. The assay is intended for laboratory professional use only.

Related assays

ViennaLab supplies the same RealFast format for HLA-B*15:02 (REF 7-630 and 7-633), HLA-A*31:01 (REF 7-640 and 7-643) and LCT -13910C>T (REF 7-150 and 7-153). For HLA-B27 by reverse hybridisation instead of real-time PCR, the HLA-B27 StripAssay is available.

• RealFast 2x Genotyping Mix (white cap; 1000 µl for REF 7-620, 320 µl for REF 7-623)
• HLA-B27 Assay Mix (purple cap, 550 µl)
• HLA-B27 Positive Control (green cap, 75 µl)
• HLA-B27 Negative Control (red cap, 75 µl)
• Instructions for Use

REF 7-620 covers 100 reactions and REF 7-623 covers 32 reactions, each in a final volume of 20 µl. The 2x Genotyping Mix contains hot-start Taq DNA polymerase and dNTPs in an optimised buffer system, so no separate enzyme, buffer or primer vial is supplied. The Assay Mix holds the gene-specific primers and the dual-labelled probes for HLA-B*27 and for the control gene. DNA extraction reagents are not supplied; ViennaLab recommends the GENXTRACT Blood DNA Extraction System (REF 2-014) or the Spin Micro DNA Extraction Kit (REF 2-020), to be ordered separately.