Journal of Virological Methods 137 (2006) 298–303 A novel accurate ACRS-PCR method with a digestion internal control for identification of wild type and YMDD mutants of hepatitis B virus strains Seyed Younes Hosseini a , Farzaneh Sabahi a,∗ , Samad Amini-Bavil-Olyaee b , Seyed-Moayed Alavian c , Shahin Merat d a Virology Department, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran b Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran c Baqiyatallah University of Medical Sciences, Tehran Hepatitis Center, Vesal Avenue, Tehran, Iran d Digestive Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran Received 7 January 2006; received in revised form 29 June 2006; accepted 4 July 2006 Available online 8 September 2006 Abstract As a consequence of the point mutation in the YMDD motif of the hepatitis B virus (HBV) polymerase gene, lamivudine-resistant mutants have been reported in chronic hepatitis B patients who underwent lamivudine therapy. The objective of the study was to develop a novel accurate artificially created restriction site (ACRS) method with a digestion internal control for identification of YMDD, YIDD and YVDD HBV strains. Three conserved, specific and diagnostic primers introducing NdeI, SspI and AleI cleavage sites were designed in order to identify YMDD, YIDD and YVDD strains, respectively; while, their reverse primers also modified with the above recognition sites in order to enzyme correctness monitoring and false outcome avoiding. Thirty-two chronic hepatitis B patients who had taken lamivudine for 1–3 years and checked by the Inno-LiPA HBV DR kit, were evaluated by the ACRS method and then compared to sequencing data. The results of the ACRS method revealed the YMDD mutant strain in 20 patients, YMDD plus YIDD pattern in 1 patient, YMDD plus YVDD in 4 patients, the YIDD in 4 patients and mixed infection with each three strains in 1 patient. The sequencing and Inno-LiPA results were in agreement with the ACRS results. The novel ACRS method is a reliable, rapid and a cost-effective technique for determination of HBV strains with the wild type and YMDD mutant patterns. © 2006 Published by Elsevier B.V. Keywords: Hepatitis B virus; Drug-resistance; YMDD motif; ACRS-PCR; Iranian patients 1. Introduction Hepatitis B virus (HBV) infection is a global health problem. Current estimates are that over 360 million people are chronic carriers of hepatitis B virus worldwide, resulting in more than 520,000 deaths annually (Kane, 1995; Lee, 1997). To date, therapy of chronic hepatitis B has remained a major clinical challenge. The development of specific inhibitors belonging to the class of nucleoside analogues that inhibit the HBV reverse transcriptase (RT) provided new therapy of this disease. Lamivu- dine, a dideoxycytidine analogue, was developed and licensed because of its antiviral effects, excellent profile of safety and tolerability, which is accompanied by improvement of liver histology (Zoulim, 2003). However, because of the HBV repli- ∗ Corresponding author. Fax: +98 21 88013030. E-mail address: sabahi f@modares.ac.ir (F. Sabahi). cation strategy and spontaneous genome variability, long-term treatment with lamivudine is associated with the selection of drug resistant mutants (Locarnini, 2003; Summers and Mason, 1982). The selection of lamivudine resistance mutants is the main concern with HBV treatment. The major site of mutation is the methionine residue in the YMDD motif (Tyrosine-Methionine-Aspartate-Aspartate) of the HBV RT gene. The mutants are characterized by a change of methionine to valine or isoleucine (YMDD to YVDD/YIDD) (Tipples et al., 1996; Allen et al., 1998). Biochemical and viro- logical breakthroughs are observed usually after the appear- ance of resistance. Deterioration of hepatic function is observed after breakthrough during lamivudine therapy, in both HBeAg positive and negative patients (Papatheodoridis et al., 2002). Monitoring of patients for the emergence of YMDD mutants during therapy can help better clinical management. The resis- tant mutant must be confirmed by the detection of mutation within the YMDD motif to help a successful treatment during 0166-0934/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jviromet.2006.07.008