HbS-HbC mpx PCR Assay

Introduction

Sickle cell disease (SCD) is a monogenic red blood cell disorder, where normal hemoglobin (HbA) is replaced by sickle hemoglobin (HbS). SCD is inherited as an autosomal codominant trait. Common types of SCD include homozygous hemoglobin SS disease, hemoglobin SC disease, and sickle beta-thalassemia. Globally, there are approximately 300,000–400,000 newborns with SCD, with over 75% of them in Africa. The World Health Organization (WHO) has estimated that SCD may contribute up to 16% of under-five mortality in some high-burden African countries where 20–30% of the population has sickle cell trait (SCT) and 1–2% of infants have SCD. Apart from HbS, there are other Hb variants (non-S sickling Hb variants) that cause sickle cell disease.

Storage and Stability

HbS-HbC mpx RealFast™ Assay is shipped on cooling blocks. On arrival, store the kit at -30°C to -15°C. Alternatively, store at 2°C to 8°C for short-term use within one month. The kit withstands up to 20 freeze/thaw cycles with no loss of activity. Avoid prolonged exposure to intense light. If stored correctly, the kit will retain full activity until the expiration date indicated on the label.

Principle of the Test

The test is based on the fluorogenic 5’ nuclease assay, also known as TaqMan® assay. Each reaction contains gene-specific primer pairs that amplify an 85 bp fragment of the HBB gene, which also serves as the PCR control. Further components are two dual-labeled, gene-specific hydrolysis probes that hybridize to the target sequence of the corresponding fragment. The proximity of the 5’-fluorescent reporter and 3’-quencher dye on intact probes prevents the reporter from fluorescing. During the extension phase of PCR the 5’ – 3’ exonuclease activity of the Taq DNA polymerase cleaves the 5’-fluorescent reporter from the hybridized probe. The physical separation of the fluorophore from the quencher dye generates a fluorescent signal in real time, which is proportional to the accumulated PCR product.

In samples positive for HBB, the Cy5.5-labeled HBB probe binds to the gene fragment and a strong fluorescence signal is detected in the Cy5.5 channel (711 nm). In samples positive for HbC, (HBB c.19G>A) the HEX-labeled HbC probe binds to the gene fragment and a strong fluorescence signal is detected in the HEX channel (556 nm). In samples positive for HbS (HBB c.20A>T), the Cy5-labeled HbS probe binds to the gene fragment and a strong fluorescence signal is detected in the Cy5 channel (670 nm). In samples negative for HbC and HbS only the Cy5.5-labeled HBB control probe hybridizes to the HBB gene fragment. Accordingly, a strong fluorescence signal is detected in the Cy5.5 channel and no or only baseline signals in the HEX and Cy5 channels. In samples compound heterozygote for HbC and HbS, strong fluorescence signals are detected in the HEX and Cy5 channels and no or only a baseline signal in the Cy5.5 channel.

Real-time PCR Instrument Compatibility

The HbS-HbC mpx RealFast™ Assay is validated for use with the Bio-Rad CFX 96, Bio-Rad CFX Opus 96, and Applied Biosystems Quantstudio 5 real-time PCR instruments calibrated for Cy5.5, Cy5 and HEX. The HbS-HbC mpx RealFast™ Assay can be combined with SMN1 RealFast™ Assay (REF 7-700). In this case, calibration of the realtime PCR device for Cy5.5, Cy5, HEX, FAM, and ROX is required.

• AB QuantStudio 5 (Applied Biosystems)
• CFX96 (Bio-Rad)
• OPUS 96 (Bio-Rad)

The kit is supplied without PCR-grade water.

Assay Performance Specifications

• The HbS-HbC mpx RealFast™ Assay is for research use only.
• Analytical performance data: currently not available.
• Limit of detection: data not available.
• Recommended DNA concentration: 0.2 to 10 ng/µl genomic DNA.

• RealFast™ 4x LIT Mix: 1 vial, white cap, 550 µl
• HbS-HbC mpx Assay Mix: 1 vial, purple cap, 275 µl
• HBB WT-Control: 1 vial, green cap, 75 µl
• HbSC MUT-Control: 1 vial, red cap, 75 µl

The RealFast™ 4x LIT Mix comprises HotStart Taq DNA polymerase and dNTPs in an optimized buffer system. The HbS-HbC Assay Mix consists of HBB gene-specific primers and dual-labeled hydrolysis probes for HBB, HbC and HbS. A wild-type and a mutant control are supplied with the kit.

The kit contains reagents for 100 reactions in a final volume of 20 µl each.