Quantification of the aflatoxin biocontrol strain Aspergillus flavus AF36 in soil, and nuts and leaves of pistachio by real-time PCR

Plant Disease ◽  
2020 ◽  
Author(s):  
MARÍA TERESA GARCÍA LOPEZ ◽  
Yong Luo ◽  
Alejandro Ortega-Beltran ◽  
Ramon Jaime ◽  
Juan Moral Moral ◽  
...  
Keyword(s):  
Real Time ◽  
Aspergillus Flavus ◽  
Real Time Pcr ◽  
Management Strategies ◽  
Cost Savings ◽  
Aflatoxin Contamination ◽  
Conidial Suspension ◽  
Toxigenic Strains ◽  
Important Time ◽  
Biocontrol Strain

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels conditioning their sale. The strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in the orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCA) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCA is labor-intensive and time-consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold values (Cq). Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). The tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of the current study will serve to design better aflatoxin management strategies for pistachio and other crops.

Real Time PCR detection of Macrobrachium rosenbergii (de Man, 1879) larvae with emphasis to their ecology

2021 ◽  
Vol 34 ◽  
pp. 12
Author(s):  
Mahadevan Harikrishnan ◽  
Deepak Jose ◽  
B. Nidhin ◽  
K.P. Anilkumar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Monsoon Season ◽  
Management Strategies ◽  
Macrobrachium Rosenbergii ◽  
Freshwater Prawn ◽  
Larval Abundance ◽  
Larval Stages ◽  
Species Specific ◽  
De Man

Species specific identification of early larval stages of many decapod crustaceans sampled from plankton collections remains cumbersome owing to lack of distinguishable characteristics, where DNA based molecular methods provide accurate results without taxonomic ambiguities. In the present study, an attempt was made to detect temporal occurrence of early zoea of freshwater prawn Macrobrachium rosenbergii (de Man) using real-time PCR assays in polyhaline, mesohaline and oligohaline areas of a tropical positive estuary, the Vembanad lake (S. India). High caridean larval abundance could be recorded in polyhaline areas in all seasons while it could be recorded in monsoon season in mesohaline and oligohaline areas. 113 DNA isolations were successfully made from morphologically identified taxonomic units (MOTU) and SYBR Green based RT-PCR amplifications using designed primer for M. rosenbergii yielded positive detections in 38 samples (34%) representing all seasons in all three zones. Positive detections could be recorded in all months except May in mesohaline areas and differed significantly (F = 17.2 p < 0.01) with the same in polyhaline and oligohaline areas. The present results of molecular detection of M. rosenbergii larvae extend confirmation of its breeding ground in Vembanad lake where appropriate management strategies could be enforced for stock conservation of this species.

scholarly journals Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 757
Author(s):  
Sara Franco Ortega ◽  
Ilenia Siciliano ◽  
Simona Prencipe ◽  
Maria Lodovica Gullino ◽  
Davide Spadaro
Keyword(s):  
Food Safety ◽  
Real Time ◽  
Aspergillus Flavus ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Screening Method ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Efficacy of a Biopesticide for Control of Aflatoxins in Corn

2010 ◽  
Vol 73 (3) ◽  
pp. 495-499 ◽  
Author(s):  
JOE W. DORNER
Keyword(s):  
Aspergillus Flavus ◽  
Aflatoxin Contamination ◽  
Naturally Occurring ◽  
Application Rates ◽  
Close Proximity ◽  
The Mean ◽  
Toxigenic Strains ◽  
And Control ◽  
Mean Concentration

A 2-year study was carried out to determine the efficacy of a biopesticide in reducing aflatoxin contamination in corn. The biopesticide, afla-guard, delivers a nontoxigenic strain of Aspergillus flavus to the field where it competes with naturally occurring toxigenic strains of the fungus. Afla-guard was applied to entire fields in two areas of Texas at either 11.2 or 22.4 kg/ha. Specific nontreated fields in close proximity to treated fields were designated as controls. Samples of corn were collected at harvest and analyzed for aflatoxins and density of toxigenic and nontoxigenic isolates of A. flavus. Aflatoxin concentrations were generally quite low in 2007, but the mean concentration in treated samples (0.5 ppb) was reduced by 85% compared with controls (3.4 ppb). In 2008, samples from treated and control fields averaged 1.5 and 12.4 ppb, respectively, an 88% reduction. There were no significant differences between the two afla-guard application rates. In conjunction with the reductions in aflatoxin contamination, treatments produced significant reductions in the incidence of toxigenic isolates of A. flavus in corn.

scholarly journals Detection and Characterization of Carboxylic Acid Amide-Resistant Plasmopara viticola in China Using a TaqMan-MGB Real-Time PCR

Plant Disease ◽  
2020 ◽  
Vol 104 (9) ◽  
pp. 2338-2345
Author(s):  
Xiaoqing Huang ◽  
Xina Wang ◽  
Fanfang Kong ◽  
Theo van der Lee ◽  
Zhongyue Wang ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Fungicide Resistance ◽  
Management Strategies ◽  
Acid Amide ◽  
Plasmopara Viticola ◽  
Carboxylic Acid Amide ◽  
Caa Fungicides

Grape production is increasing globally and so are problems with downy mildew, one of the main constraints in grape production. Downy mildew on grape is caused by Plasmopara viticola, an obligate biotrophic pathogen belonging to the oomycetes. Control of the disease is usually performed by fungicide applications, of which carboxylic acid amide (CAA) fungicides represent one of the most widely used groups of fungicides. Our previous research showed that the extensive application of CAA fungicides can result in fungicide resistance and in China, CAA-resistant isolates of P. viticola were collected from the field in 2014. To monitor the distribution and spread of CAA fungicide resistance, we developed a TaqMan-minor groove binder (MGB) real-time PCR-based method designed on a functional mutation in the PvCesA3 gene that allows efficient identification of CAA fungicide resistant and sensitive genotypes. The assay was validated on 50 isolates using Sanger sequencing and fungicide bioassays and exploited in a comprehensive survey comprising 2,227 single-sporangiophore isolates from eight major grapevine regions in China. We demonstrate that CAA fungicide resistance in P. viticola is widespread in China. On average, 53.3% of the isolates were found to be resistant, but marked differences were found between locations with percentages of resistant isolates varying from 0.3 to 96.6%. Furthermore, the frequency of CAA-resistant isolates was found to be significantly correlated with the exposure to CAA fungicides (P < 0.05). We further discussed the possibilities to apply the TaqMan-MGB real-time PCR assay to assess the frequency of fungicide-resistant P. viticola isolates in each region or vineyard, which would facilitate the correct choice of fungicide for grape downy mildew and resistance management strategies.

scholarly journals Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction

2007 ◽  
Vol 56 (6) ◽  
pp. 766-771 ◽  
Author(s):  
Zheng-Jiang Cheng ◽  
Li-Hua Hu ◽  
Wen-Rong Fu ◽  
Yi-Rong Li
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Cost Savings ◽  
Serum Samples ◽  
Hepatitis B Virus Dna ◽  
B Virus ◽  
High Level

The purpose of this study was to quantify hepatitis B virus DNA by direct real-time PCR from serum without the need for DNA extraction. Crossing point (Cp) values were determined automatically using the second derivative maximum mode. Since serum samples from patients are inevitably haemolysed, lipaemic or icteric, the interference of endogenous substances from the serum in real-time PCR was evaluated. The result showed that, although serum protein quenched the intensity of fluorescence, the Cp value adopted to calculate the quantity of DNA copies remained unchanged. Importantly, real-time PCR from serum with or without DNA extraction reached a high level of concordance. This direct serum PCR method without the DNA extraction and gel electrophoresis allows for substantial labour and cost savings. In addition, it is also suitable for rapid DNA quantification during clinical diagnosis.

A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food

2016 ◽  
Vol 79 (5) ◽  
pp. 810-815 ◽  
Author(s):  
FEREIDOUN FORGHANI ◽  
SHUAI WEI ◽  
DEOG-HWAN OH
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
High Resolution ◽  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Cost Savings ◽  
High Resolution Melt ◽  
And Staphylococcus Aureus

ABSTRACTThree important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 103 CFU/g without an enrichment step and 3.7 × 101 CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus.

Evaluation of a Real time polymerase chain reaction assay for the detectionof aflatoxin / sterigmatocystin producing strains of Aspergillus spp.

2017 ◽  
Author(s):  
Paviter Kaur ◽  
R. Singh ◽  
T. S. Rai ◽  
N. S. Sharma ◽  
A. . Arora
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Aflatoxin Biosynthesis ◽  
Chain Reaction ◽  
Biosynthesis Gene ◽  
Food And Feed ◽  
Polymerase Chain ◽  
Toxigenic Strains ◽  
Feed Samples

Aflatoxin and sterigmatocystin produced by Aspergillus flavus, A. parasiticus and A. versicolor are common mycotoxins found in food and feed stuffs. Sterigmatocystin is a direct precursor in aflatoxin biosynthesis. In the present study, Real time PCR using TaqMan probe chemistry was standardized for amplification of the aflatoxin/sterigmatocystin biosynthesis gene omt-1 of Aspergillus spp. The gene was amplified in six cultures of Aspergillus spp. isolated from 53 aflatoxin positive feed samples hence indicating their aflatoxigenic potential. Data clearly revealed that the real time PCR technique is efficient in distinguishing toxigenic strains of A. parasiticus, A. versicolor and A. flavus from other molds commonly inhabiting the feed.

scholarly journals Prevalence of Aflatoxin Contamination in Maize and Groundnut in Ghana: Population Structure, Distribution, and Toxigenicity of the Causal Agents

Plant Disease ◽  
2018 ◽  
Vol 102 (4) ◽  
pp. 764-772 ◽  
Author(s):  
D. Agbetiameh ◽  
A. Ortega-Beltran ◽  
R. T. Awuah ◽  
J. Atehnkeng ◽  
P. J. Cotty ◽  
...  
Keyword(s):  
Population Structure ◽  
Open Access ◽  
Aspergillus Flavus ◽  
Economic Burden ◽  
Management Strategies ◽  
Aflatoxin Contamination ◽  
Agroecological Zones ◽  
First Time ◽  
Open Access Article ◽  
Aspergillus Section

Aflatoxin contamination in maize and groundnut is perennial in Ghana with substantial health and economic burden on the population. The present study examined for the first time the prevalence of aflatoxin contamination in maize and groundnut in major producing regions across three agroecological zones (AEZs) in Ghana. Furthermore, the distribution and aflatoxin-producing potential of Aspergillus species associated with both crops were studied. Out of 509 samples (326 of maize and 183 of groundnut), 35% had detectable levels of aflatoxins. Over 15% of maize and 11% of groundnut samples exceeded the aflatoxin threshold limits set by the Ghana Standards Authority of 15 and 20 ppb, respectively. Mycoflora analyses revealed various species and morphotypes within the Aspergillus section Flavi. A total of 5,083 isolates were recovered from both crops. The L morphotype of Aspergillus flavus dominated communities with 93.3% of the population, followed by Aspergillus spp. with S morphotype (6%), A. tamarii (0.4%), and A. parasiticus (0.3%). Within the L morphotype, the proportion of toxigenic members was significantly (P < 0.05) higher than that of atoxigenic members across AEZs. Observed and potential aflatoxin concentrations indicate that on-field aflatoxin management strategies need to be implemented throughout Ghana. The recovered atoxigenic L morphotype fungi are genetic resources that can be employed as biocontrol agents to limit aflatoxin contamination of maize and groundnut in Ghana. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

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