Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI

Abstract

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.

Identification of Aspergillus species in feed fed to caged birds using morphological characteristics in Zaria, Nigeria

“…This study agreed with various reports that have been published that used of morphological characteristics as a key factor for the identification of fungi (Bandh et al, 2012). The features of Aspergillus species isolated in this study conform to different Aspergillus species enumerated in standard text (Raper and Fennell, 1965;Domsch et al, 1980;Samson and Pitt, 2000;McClenny, 2005;Diba et al, 2007;Gams et al, 1985;Ellis et al, 2007). The result of this study showed that there was a high level of Aspergillus contamination (96.8%) in poultry feed, fed to cage birds, these could be as a result of the ingredient in the feed which agrees with Aly and Anwer (2009), that Aspergillus species are major contaminant of grain used for poultry feed.…”

Aspergillus species as emerging causative agents of onychomycosis

“…Instead, the phenomenon of aging led to an increase in some disorders such as peripheral vascular disease of nails, diabetes, repeated nail trauma, and immune function disorders, which are all noted to be risk factors of nail infections by molds [6,23]. The high frequency of nail infections due to A. flavus can be attributed to the fact that this species is the most frequent Aspergillus isolated from the environment in Iran [9] and this species is one of the common pathogenic fungi to humans [19].…”

PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus

“…Also, in ITS-RFLP designed by Diba et al (2014), it was almost impossible to discriminate between the two clinically important species, A. fumigatus and A. flavus. But in our investigation it was shown that the digestion of Beta tubulin with AlwI generated bands specific for A. fumigatus and A. flavus (Fig 1).…”

“…Given that DNA sequencers are still not widely available in clinical laboratories, several alternative methods have been used to distinguishing Aspergillus species, including nested polymerase chain reaction (PCR) (Zhao et al, 2001), RFLP (Restriction Fragment Length Polymorphism) (Diba et al, 2007;Diba et al, 2014), SSCP (Single-Strand Conformation Polymorphism) (Diba et al, 2008), and RAPD-PCR (Random Amplified Polymorphic DNA) (Raclasky et al, 2006). However, the majority of these procedures have never gained general acceptance due to various limitations, especially their limited discriminatory power to differentiate clinically relevant Aspergillus species.…”

“…of the most common Aspergillus species.Soleiro et al, examined the variation within gliotoxin producer strains of A. fumigatus using three enzymes BccI, MspI and Sau3AI and found band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers(Soleiro et al, 2013) Diba et al (2014). by single restriction enzyme MwoI on ITS regions of rDNA gene developed a method for species identification of several clinically significant Aspergillus including A. flavus,A.…”