PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus.
Aspergillus species are important agents of life-threatening infections in immunosuppressed patients. Proper speciation in the Aspergilli has been justified based on varied fungal virulence, clinical presentations, and antifungal resistance. Accurate identification of Aspergillus species usually relies on fungal DNA sequencing but this requires expensive equipment that is not available in most clinical laboratories. We developed and validated a discriminative low-cost PCR-based test to discriminate Aspergillus isolates at the species level. The Beta tubulin gene of various reference strains of Aspergillus species was amplified using the universal fungal primers Bt2a and Bt2b. The PCR products were subjected to digestion with a single restriction enzyme AlwI. All Aspergillus isolates were subjected to DNA sequencing for final species characterization. The PCR-RFLP test generated unique patterns for six clinically important Aspergillus species, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus and Aspergillus nidulans. The one-enzyme PCR-RFLP on Beta tubulin gene designed in this study is a low-cost tool for the reliable and rapid differentiation of the clinically important Aspergillus species.
Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
Tuba
T
Nasri
Invasive Fungi Research Center (IFRC), Department of Medical Mycology and Parasitology/School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Mohammad Taghi
MT
Hedayati
Invasive Fungi Research Center (IFRC), Department of Medical Mycology and Parasitology/School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Mahdi
M
Abastabar
Molecular Biology Laboratory, Santa Casa de Misericórdia de Porto Alegre, Porto Alegre, Brazil; Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, Brazil.
Alessandro C
AC
Pasqualotto
Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
Mojtaba Taghizadeh
MT
Armaki
Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
Akbar
A
Hoseinnejad
Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.
Mojtaba
M
Nabili
D001228:Aspergillosis / Q000382:microbiology; D001230:Aspergillus / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004783:Environmental Microbiology; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics*; D014404:Tubulin / Q000235:genetics*
D004271:DNA, Fungal; D014404:Tubulin
AlwI; Aspergillus; Identification; PCR-RFLP