Department of Forensic Medicine and Human Genetics, Kurume University Hospital, Kurume, Japan.
Transfusion. 2010 Jun;50(6):1322-7. doi: 10.1111/j.1537-2995.2009.02581.x. Epub 2010 Jan 22.
Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method.
A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates.
Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method.
The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).
由于缺乏血清触珠蛋白（Hp）抗体，无触珠蛋白血症患者存在发生严重过敏输血反应的风险。纯合子状态的触珠蛋白基因缺失（HP(del)）是先天性无触珠蛋白血症的唯一已知病因，在输血前进行 HP(del) 的临床诊断对于预防过敏休克至关重要。我们最近开发了一种 5'-核酸酶（TaqMan）实时聚合酶链反应（PCR）方法。
开发了一种基于 SYBR Green I 的双实时 PCR 检测法，该检测法使用两个正向引物和一个共同的反向引物，随后进行熔解曲线分析，以在单个管中确定 HP(del)的基因型。此外，为了避免初始 DNA 提取，我们检查了连续稀释的血样作为 PCR 模板。
HP(del)等位基因的等位基因鉴别在血液样本稀释度为 1:64 至 1:1024 时产生最佳结果。2231 份血样的结果与 TaqMan 实时 PCR 法完全一致。
基于 SYBR Green I 的方法检测 HP(del)等位基因的检出率与基于 TaqMan 的方法相当。由于其初始成本低，并且可以使用经济实惠的实时 PCR 仪器进行分析，因此该方法易于应用，并且适用于高通量分析，是 HP(del)等位基因鉴别替代方法。
