Development of RFLP method for rapid differentiation of Aspergillus flavus and Aspergillus oryzae, two species with high importance in clinical and food microbiology - 17/08/22

Doi : 10.1016/j.mycmed.2022.101274

Mahdi Abastabar ^a,^b,^⁎ , Shafigheh Shabanzadeh ^a,^b, Reza Valadan ^c, Sabah Mayahi ^a,^b, Iman Haghani ^a,^b, Shaghayegh Khojasteh ^a,^b, Sanaz Nargesi ^a,^b, Seyedmojtaba Seyedmousavi ^b,^d, Mohammad Taghi Hedayati ^a,^b,^⁎
^a Department of Medical Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
^b Invasive Fungi Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran
^c Molecular and Cell Biology Research Center (MCBRC), Mazandaran University of Medical Sciences, Sari, Iran
^d Microbiology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, United States

^⁎Corresponding authors.

Abstract

Aspergillus flavus and Aspergillus oryzae, two species of Aspergillus section Flavi, are of utmost significance in health, medicine, biotechnology, and foods industries. The methods currently used in mycology for the discrimination of these two closely related species were unable to definitively and rapidly distinguish. The present study aimed to develop a restriction fragment length polymorphism (RFLP) test based on the cyp51A gene to discriminate between A. flavus and A. oryzae. The cyp51A gene sequences of A. flavus and A. oryzae reference strains were amplified using the specific primers CYP51AF and CYP51AR. The polymerase chain reaction (PCR) products were subjected to digestion with a restriction enzyme, HincII. The polymerase chain reaction (PCR)- RFLP test created specific patterns for standard strains, as well as clinical and environmental A. flavus and A. oryzae isolates. The one-enzyme PCR-RFLP test on the cyp51A gene designed in the present study is a remarkably simple, reliable, and low-cost method for the accurate and rapid differentiation of A. flavus and A. oryzae isolated from clinical and environmental samples.

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Keywords : Aspergillus flavus, Aspergillus oryzae, Cyp51A, RFLP test

Plan

Introduction

Materials and methods

Fungal strains

Molecular tests

In silico analysis

DNA extraction and polymerase chain reaction amplification

Digestion of PCR products by restriction enzyme

Evaluation of length polymorphisms by electrophoresis

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Vol 32 - N° 3

Article 101274- août 2022 Retour au numéro

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Development of RFLP method for rapid differentiation of Aspergillus flavus and Aspergillus oryzae, two species with high importance in clinical and food microbiology - 17/08/22

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