Ramazan GÜNEŞAÇAR, Bekir Mehmet KELLECİ, Mehmet Yaşar ÖZKARS, İlkay ADANIR, Mustafa ÇELİK

PCR-SSP tekniğine dayalı laboratuvar yapımı HLA-B*27 optimizasyonu ve test kiti geliştirilmesi

AAmaç: Çalışmada Ankilozan spondilit ve diğer spondiloartropatilerin moleküler tanısında kullanılmak üzere güvenilir, ekonomik ve rutin kullanıma uygun laboratuvar yapımı (in-house) HLA-B*27 PCR-SSP tanı kiti geliştirilmesi amaçlanmıştır. Gereç ve Yöntem: Çalışmaya, ticari kitler kullanılarak HLA-B*27 pozitif olarak saptanan 119 ve negatif olarak saptanan 446 olmak üzere toplam 565 olgu dahil edildi. HLA-B*27 tipleme için sekansa spesifik primerler ve beta globin gen bölgesinden yararlanılarak dizayn edilen internal kontrol primer çifti kullanılarak PCR optimizasyonu gerçekleştirildi. Bulgular: HLA-B*27’si pozitif olan olgularda, %2’lik agaroz jelinde 273 bç internal kontrol bandının ilerisinde 149 bç’lik spesifik bant olmak üzere çift bant, B*27 negatif hastalarda ise sadece 273 bç’lik internal kontrol bandı saptandı. Ticari kitler kullanılarak HLA-B*27’si pozitif olarak saptanan 119 ve negatif olarak saptanan 446 bireyin sonuçları ile tarafımızdan geliştirilen PCR-SSP kiti kullanılarak elde edilen sonuçların tamamen uyumlu olması nedeniyle sensitivite ve spesifite %100 olarak belirlendi. Geliştirdiğimiz laboratuvar yapımı PCR-SSP kitinin maliyetinin, ticari kitlere göre en az 50 kat daha ucuz olduğu görüldü. Sonuç: Ticari kitler kullanılarak HLA-B*27’si pozitif olarak saptanan 119 ve negatif olarak saptanan 446 olgunun tamamında, geliştirdiğimiz yöntemle de birebir uyumlu sonuçlar elde edildi. Bu bulgular geliştirdiğimiz kitin güvenilir, ekonomik ve rutin kullanıma uygun olduğunu göstermektedir.

Anahtar Kelimeler:

HLA-B*27, PCR-SSP, Test kiti geliştirilmesi, Ankilozan spondilit, Spondiloartropatiler

In-house HLA-B*27 optimization based on PCR-SSP technique and test kit development

Purpose: In the present study, we aimed to develop a reliable, cost effective and suitable for routine use in-house HLA-B*27 PCR-SSP typing kit for the molecular diagnosis of Ankylosing spondylitis and other spondyloarthropathies. Materials and Methods: The study included 565 cases with 446 negative and 119 positive HLA-B*27 subjects. PCR optimization was performed for HLA-B*27 alleles using sequence-specific and internal control primers designed based on HLA-B and beta globin gene sequences, respectively. Results: The presence of 149 bp specific band with 273 bp internal control band showed B*27 positivity and the presence of only 273 bp control band showed B*27 negativity on 2.0% agarose gel. The HLA-B*27 results show 100% sensitivity and specificity to our in-house SSP kit developted by our laboratory and the commercially available SSP kits. The cost of the in-house PCR-SSP kit we developed was at least 50 times cheaper than the commercial kits. In addition to the faultless correlation between the kits, our in-house SSP kit did not cross-react with other HLA-B alleles such as B*07, B*12, B*13, B*16, B*17, B*22, B*37, B*40, B*41, B*42, B*47 and B*48.Conclusion: The fully evaluated findings show that in-house SSP kit for HLA-B*27 typing we developed is reliable, cost effective and suitable for routine use in tissue typing and genetic laboratories.

Keywords:

HLA-B*27, PCR-SSP, Test kit development, Ankylosing spondylitis, Spondyloarthropathies,

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