Malaysian Journal of Microbiology, Vol 19(6) Special Issue 2023, pp. xxx-xxx DOI: http://dx.doi.org/10.21161/mjm.230009 Malaysian Journal of Microbiology Published by Malaysian Society for Microbiology (In since 2011) 1 ISSN (print): 1823-8262, ISSN (online): 2231-7538 *Corresponding author Optimizing electrochemical DNA biosensors for the detection of avian infectious bronchitis virus Md. Safiul Alam Bhuiyan 1,4 , Gilbert Ringgit 1 , Zarina Amin 1 , Ag Muhammad Sagaf Abu Bakar 2 , Suryani Saallah 1 , Sharifudin Md. Shaarani 3 and Shafiquzzaman Siddiquee 1 * 1 Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia. 2 Jabatan Perkhidmatan Veterinar Sabah, Makmal Diagnosa Veterinar Kota Kinabalu, Peti Surat No 59, 89457, Tanjung Aru, Sabah, Malaysia. 3 Food Biotechnology Program, Faculty of Science and Technology, Universiti Sains Islam Malaysia, Bandar Baru Nilai, 71800 Nilai, Sembilan, Malaysia. 4 Livestock Production, Faculty of Sustainable Agriculture, Universiti Malaysia Sabah, Sandakan, Sabah, Malaysia. Email: shafiqpab@ums.edu.my ABSTRACT Aims: The aim of the study is to develop the optimised parameters of electrochemical DNA biosensors for the specific detection of the Infectious Bronchitis Virus (IBV) in chickens. The ultimate goal is to further create a highly sensitive and specific biosensor that can be used for on-site monitoring of IBV on poultry farms. Methodology and results: In this study, an electrochemical DNA biosensor was developed for detecting a specific sequence in the IBV genome. The process involved attaching a NH2-ssDNA probe to a gold electrode, followed by hybridization with the target DNA. Various parameters like buffer, pH, scan rate, incubation time, redox indicators and temperature were optimised using cyclic voltammetry. The probe DNA was designed to enhance hybridization efficiency, which was assessed by measuring current signals. The biosensor, under optimal conditions, demonstrated high sensitivity and specificity when tested with different sequences, including complementary, non-complementary and mismatched ones. Cross-reactivity studies against non-IBV viruses showed distinguishable current signals. These findings have implications for developing a portable on-site IBV monitoring device for use on farms. Conclusion, significance and impact of study: The optimised parameters and specificity of the electrochemical DNA biosensor suggest its potential for the development of a portable device for on-site monitoring of IBV on poultry farms. This device could prove to be a valuable tool for the early detection of IBV, helping to prevent further spread of the disease. However, it's essential to conduct further research to ensure the practicality and accuracy of the biosensor in real-world farm settings. Keywords: Avian infectious bronchitis virus, cyclic voltammetry, electrochemical biosensor, hybridization, immobilisation INTRODUCTION Infectious bronchitis virus (IBV) is an avian coronavirus of the Coronaviridae family that has the greatest impact on broiler, breeder and layer production worldwide. Based on genome sequencing, IBV belongs to the gamma- coronavirus and only some numbers are in Delta-CoV (de Wit and Cook, 2020) genus, which is essential for understanding its genetic relationship to other coronaviruses (Wickramasinghe et al., 2011; Sharma et al., 2020). However, beta- and gamma-coronaviruses (CoV) carry an open reading frame (ORF) that encodes the structural proteins in the 3'-terminal and the non- structural proteins in the 5'-terminal regions of the genome, which bind to the host cell as distinct receptors for pathogenesis. IBV has led to economic losses in the poultry industry as the number of local IBV variants has increased due to frequent genetic mutations, replications and gene deletions. When infected with IBV, the chicken becomes immunosuppressed and quickly becomes infected with secondary bacterial infections. This therefore poses a major challenge for the prevention and control of IBV (Jackwood, 2012; Bhuiyan et al., 2021). IBV variants or strains can be mutually protective and response to vaccines ranges from very low to moderate, which can lead to an outbreak of IBV in vaccinated flocks (Xu et al., 2007; Xu et al., 2015). Between 1990 and 2020, studies on IBVs were conducted across various states in Malaysia, including Sabah where the predominant IBV strain was identified as the IBV QX-like variant (47%) followed by the Malaysia Variants (13%) and vaccine