Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Sep;12(3):1628-1632.
doi: 10.3892/etm.2016.3479. Epub 2016 Jun 24.

Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

Majid Zarrin  1 , Maryam Erfaninejad  2
Affiliations

Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

Majid Zarrin et al. Exp Ther Med. 2016 Sep.

Abstract

Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates.

Keywords: Aspergillus flavus; internal transcribed spacer rDNA region; polymerase chain reaction-restriction fragment length polymorphism.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Internal transcribed spacer (ITS) regions of Aspergillus flavus isolates were amplified by polymerase chain reaction using ITS1 and ITS4 primers and the products were separated by agarose gel electrophoresis. M, 100 bp ladder; lane 1, Kh4 isolate; lane 2, Kh5 isolate; lane 3, Kh6 isolate; lane 4, Kh9 isolate; lane 5, Kh10 isolate; lane 6, Kh11 isolate; lane 7, M25 isolate; lane 8, M26 isolate; lane 9, M27 isolate; lane 10, M28 isolate; lane 11, M29 isolate; lane 12, M32 isolate; lane 13, M33 isolate; lane 14, no template control.
Figure 2.
Figure 2.
Restriction fragment pattern of internal transcribed spacer (ITS) polymerase chain reaction (PCR) products of Aspergillus flavus digested with EcoRI. Lane M1, 100 bp ladder; lane 1, Z7 isolate; lane 2, Z8 isolate; lane 3, Z9 isolate; lane 4, Z10 isolate; lane 5, PFCC101 isolate; lane 6, PFCC126 isolate; lane 7, PFCC159 isolate; lane 8, PFCC209 isolate; lane 9, PFCC170 isolate; lane 10, PFCC173 isolate; lane 11, undigested ITS PCR product; lane M2, 1 kb ladder.
Figure 3.
Figure 3.
Restriction fragment pattern of internal transcribed spacer (ITS) polymerase chain reaction (PCR) products of Aspergillus flavus digested with TaqI. Lane M, 100 bp ladder; lane 1, Z9 isolate; lane 2, Z10 isolate; lane 3, D1 isolate; lane 4, D2 isolate; lane 5, M29 isolate; lane 6, M32 isolate; lane 7, M33 isolate; lane 8, undigested ITS PCR product.
Figure 4.
Figure 4.
Restriction fragment pattern of internal transcribed spacer (ITS) polymerase chain reaction (PCR) products of Aspergillus flavus digested with HaeIII. Lane M1, 100 bp ladder; lane 1, M2 isolate; lane 2, M4 isolate; lane 3, Kh1 isolate; lane 4, Kh2 isolate; lane 5, Kh4 isolate; lane 6, Kh5 isolate; lane 7, Kh6 isolate; lane 8, Kh8 isolate; lane 9, undigested ITS PCR product; lane M2, 100 bp ladder.
Figure 5.
Figure 5.
Restriction fragment pattern of internal transcribed spacer (ITS) polymerase chain reaction (PCR) products of Aspergillus flavus digested with HaeIII. Lane M1, 100 bp ladder; lane 1, M1 isolate; lane 2, M3 isolate; lane 3, Kh3 isolate; lane 4, Kh7 isolate; lane 5, Z9 isolate; lane 6, Z10 isolate; lane 7, undigested ITS PCR product; lane M2, 100 bp ladder.
See this image and copyright information in PMC

References

    1. Krishnan S, Manavathu EK, Chandrasekar PH. Aspergillus flavus: An emerging non-fumigatus Aspergillus species of significance. Mycoses. 2009;52:206–222. doi: 10.1111/j.1439-0507.2008.01642.x. - DOI - PubMed
    1. Pasqualotto AC. Differences in pathogenicity and clinical syndromes due to Aspergillus fumigatus and Aspergillus flavus. Med Mycol. 2009;47(Suppl 1):S261–S270. doi: 10.1080/13693780802247702. - DOI - PubMed
    1. Ehrlich KC, Mack BM. Comparison of Expression of Secondary Metabolite Biosynthesis Cluster Genes in Aspergillus flavus, A. parasiticus, and A. oryzae. Toxins (Basel) 2014;6:1916–1928. doi: 10.3390/toxins6061916. - DOI - PMC - PubMed
    1. Karthikeyan M, Sandosskumar R, Mathiyazhagan S, Mohankumar M, Valluvaparidasan V, Kumar S, Velazhahan R. Genetic variability and aflatoxigenic potential of Aspergillus flavus isolates from maize. Arch Phytopathol Plant Protect. 2009;42:83–91. doi: 10.1080/03235400600950961. - DOI
    1. Zhang ZG, Zhang JY, Zheng XB, Yang YW, Ko WH. Molecular distinctions between Phytophthora capsici and P. tropicalis based on ITS sequences of ribosomal DNA. J Phytopathol. 2004;152:358–364. doi: 10.1111/j.1439-0434.2004.00856.x. - DOI

LinkOut - more resources