Investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR, based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients Mohammad Amin Honardoost a,1 , Hosein Tabatabaeian b,1 , Mojtaba Akbari c , Mansoor Salehi c,d, ⁎ a Division of Cellular and Molecular Biology, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran b Genetics Division, Biology Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran c School of Medicine, Isfahan University of Medical Science, Isfahan, Iran d Medical Genetics Center of Genome, Shariati St., Isfahan, Iran abstract article info Article history: Received 11 November 2013 Received in revised form 11 May 2014 Accepted 30 May 2014 Available online xxxx Keywords: Beta thalassemia Conventional ARMS PCR Sequencing Tetra primer ARMS PCR Purpose: Beta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra prim- er ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients. Methods: Fifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were ana- lyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing. Results: All three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results. Conclusion: Tetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is sug- gested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR meth- od. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Beta-thalassemia syndromes refer to a category of hemoglobin ab- normalities caused by mutations in the b-globin gene. These various mutations can result in reduction or loss of b-globin chain synthesis, called β + mutations and β 0 mutations respectively (Galanello and Origa, 2010). Beta-thalassemia, a monogenic autosomal recessive dis- ease, is comparatively prevalent worldwide, but it is regarded rampant in the Middle East, especially Iran. The global average associated with car- riers of beta-thalassemia is close to 3%, while it is 5% for Iranian population (Najmabadi et al., 2001). The accurate detection and screening of high- risk couples could lead to improvements of the beta-thalassemia com- plications. So far, approximately 200 different mutations in the divers regions of the b-globin gene have been reported which are related to onset of the disease (Giardine et al., 2007). Almost 20 different mutations are introduced as common mutations in Iran, with various incidence rates in cities across the country (Derakhshandeh-Peykar et al., 2008; Najmabadi et al., 2001). The frequency of common mutations in Iran was first identified by Najmabadi et al. as the following statistics: 34% for IVS-II-I, 4.76% Gene xxx (2014) xxx–xxx Abbreviations: PCR, polymerase chain reaction; RFLP, restriction fragment length poly- morphism; ARMS, amplification refractory mutation system; T-ARMS, Tetra primer ARMS. ⁎ Corresponding author at: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan 81744-176, Iran. E-mail addresses: mo.honardost@yahoo.com (M.A. Honardoost), Hosein.tabatabaeian@gmail.com (H. Tabatabaeian), m_akbari@med.mui.ac.ir (M. Akbari), m_salehi@med.mui.ac.ir (M. Salehi). 1 Co-first authors. GENE-39722; No. of pages: 6; 4C: http://dx.doi.org/10.1016/j.gene.2014.05.071 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Please cite this article as: Honardoost, M.A., et al., Investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.071