scholarly journals PCR Detection of Aspergillus flavus Isolates for Aflatoxin B1 producer

2013 ◽  
Vol 7 (3) ◽  
pp. 81-89
Author(s):  
Abdulkareem Jasim Hashim ◽  
Abdulkareem A. Al-Kazaz ◽  
Hadeel Waleed Abdulmalek
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Pure Culture ◽  
Regulatory Gene ◽  
Rapid Assessment ◽  
Chain Reaction ◽  
Good Producer ◽  
Culture Systems ◽  
Polymerase Chain

The ability of five Aspergillus flavus that produce Aflatoxin B1 have been detected using coconut medium as substrate. Chromatographical analysis by TLC and HPLC revealed that, three out of five isolates were a good producer for the Aflatoxin B1. In this study, rapid assessment of five isolates of A. flavus was accomplished using an indigenously designed primer pair for the Aflatoxin regulatory gene aflR in polymerase chain reaction (PCR). Specificity was assayed in pure culture systems using DNA extracted from five different A. flavus isolates as PCR template. Positive amplification was achieved only with DNA from A. flavus that produce Aflatoxin B1.

scholarly journals Identification of aflatoxigenic fungi using polymerase chain reaction-based assay

2014 ◽  
pp. 259-269 ◽  
Author(s):  
Vladislava Soso ◽  
Marija Skrinjar ◽  
Nevena Blagojev ◽  
Slavica Veskovic-Moracanin
Keyword(s):  
Polymerase Chain Reaction ◽  
Regulatory Gene ◽  
Control Point ◽  
Complex Mixture ◽  
Target Sequence ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Close Link ◽  
Critical Control Point ◽  
Polymerase Chain

As the aflatoxins represent a health-risk for humans because of their proven carcinogenicity, food-borne fungi that produce them as secondary metabolites, mainly Aspergillus flavus and Aspergillus parasiticus, have to be isolated and identified. The best argument for identifying problem fungi is that it indicates control points within the food system as part of a hazard analysis critical control point (HACCP) approach. This assumes there is a close link between fungus and toxin. Conventional methods for isolation and identification of fungi are time consuming and require admirably dedicated taxonomists. Hence, it is imperative to develop methodologies that are relatively rapid, highly specific and as an alternative to the existing methods. The polymerase chain reaction (PCR) facilitates the in vitro amplification of the target sequence. The main advantages of PCR is that organisms need not be cultured, at least not for a long time, prior to their detection, target DNA can be detected even in a complex mixture, no radioactive probes are required, it is rapid, sensitive and highly versatile. The gene afl-2 has been isolated and shown to regulate aflatoxin biosynthesis in A. flavus. Also, the PCR reaction was targeted against aflatoxin synthesis regulatory gene (aflR1) since these genes are nearly identical in A. flavus and A. parasiticus in order to indicate the possibility of detection of both the species with the same PCR system (primers/reaction). [Projekat Ministarstva nauke Republike Srbije, br. III46009] <br><br><font color="red"><b> This article has been retracted. Link to the retraction <u><a href="http://dx.doi.org/10.2298/APT1647265E">10.2298/APT1647265E</a><u></b></font>

scholarly journals Detection the Ability of Aspergillus flavus Isolated from Wheat Grains for Aflatoxin B1 Production using RT- PCR

2015 ◽  
Vol 9 (2) ◽  
pp. 102-106
Author(s):  
Eman Noaman Ismail
Keyword(s):  
Molecular Weight ◽  
Triticum Aestivum ◽  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Reverse Transcription ◽  
Regulatory Gene ◽  
Rapid Assessment ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Wheat Grains

Aspergillus flavus is aflatoxinogenic and potential aflatoxins producers in agriculturalcommodities. The present study was conducted determine the ability of eleven strains of A. flavusisolated from Iraqi wheat grains Triticum aestivum. The isolates have been detected by molecularmethods using Reverse Transcriptase RTPCR. In this study, RNA was extracted from A. flavus,cDNA synthesis and rapid assessment of eleven isolates of A. flavus was accomplished usingprimer pair for the aflatoxin regulatory gene aflR Reverse transcription-Polymerase chainreaction (RT–PCR).Positive amplification was achieved for all the isolates with a molecularweight 798 to aflR1 and 400bp to aflR2. Also the result of the amplification showed there are nodifferences with the two molecular weight between the 11 isolated strains of A. flavus in theiraflatoxin B1 production, but the first strain differed in their banding florescence as comparedwith others strains this reflect the genetic differences in aflatoxin B1 production between them.

Polymerase Chain Reaction–Mediated Characterization of Molds Belonging to the Aspergillus flavus Group and Detection of Aspergillus parasiticus in Peanut Kernels by a Multiplex Polymerase Chain Reaction

2002 ◽  
Vol 65 (5) ◽  
pp. 840-844 ◽  
Author(s):  
RUEY-SHYANG CHEN ◽  
JWU-GUH TSAY ◽  
YU-FEN HUANG ◽  
ROBIN Y.-Y. CHIOU
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Flavus ◽  
Aspergillus Parasiticus ◽  
Regulatory Protein ◽  
Aflatoxin Production ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Target Dna ◽  
Polymerase Chain ◽  
Aspergillus Flavus Group

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxi-genicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

DETECTION OF SYSTEMIC DONOR INFECTION AND ALLOGRAFT CONTAMINATION USING POLYMERASE CHAIN REACTION AND RAPID SHAKING CULTURE SYSTEMS.

2000 ◽  
Vol 69 (Supplement) ◽  
pp. S386
Author(s):  
Hitomi Sasaki ◽  
Kiyotaka Hoshinaga ◽  
Kiyohito Ishikawa ◽  
Toru Higuchi ◽  
Shintaro Mori ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Culture Systems ◽  
Polymerase Chain ◽  
Allograft Contamination

scholarly journals Aspergillus flavus Biomass in Maize Estimated by Quantitative Real-Time Polymerase Chain Reaction Is Strongly Correlated with Aflatoxin Concentration

Plant Disease ◽  
2009 ◽  
Vol 93 (11) ◽  
pp. 1163-1170 ◽  
Author(s):  
Santiago X. Mideros ◽  
Gary L. Windham ◽  
W. Paul Williams ◽  
Rebecca J. Nelson
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Aspergillus Flavus ◽  
Fungal Biomass ◽  
Chain Reaction ◽  
Local Conditions ◽  
Aflatoxin Concentration ◽  
Aflatoxin Accumulation ◽  
Polymerase Chain ◽  
Maize Kernels

Aspergillus flavus causes ear rot of maize and produces aflatoxins that can contaminate grain even in the absence of visible symptoms of infection. Resistance to aflatoxin accumulation and pathogen colonization are considered distinct traits in maize. Colonization of grain by fungi such as A. flavus has been difficult to quantify. We developed and validated two quantitative real-time polymerase chain reaction (qPCR) assays to estimate fungal biomass in maize tissues. In order to study the relationship between fungal biomass and aflatoxin accumulation, qPCR was conducted and aflatoxin concentrations were assayed in milled samples of mature maize kernels for two diverse sets of maize germplasm. The first was a set of hybrids that was inoculated with A. flavus in a conducive field environment in Mississippi. These hybrids, mainly early tropical and non-stiff-stalk genotypes adapted to local conditions, carry known sources of resistance among their progenitors. The second set, also tested in Mississippi, was a group of inbred lines representing a wider sample of maize genetic diversity. For both sets, our results showed a high correlation between fungal load and aflatoxin concentration in maize kernels. Our qPCR methodology could have a direct impact on breeding programs that aim to identify lines with resistance to aflatoxin accumulation, and set the stage for future studies on the genetic dissection of aflatoxin-related traits.

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