Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/17469
Title: Aflatoxin Biosynthesis Gene Profiling and Biological Control of Aflatoxin M1
Authors: Usman, Muhammad
Keywords: Biological & Medical Sciences
Biotechnology
Issue Date: 2019
Publisher: Government College University, Faisalabad
Abstract: Aflatoxins have been known to badly affect humans and livestock through immunosuppression, carcinogenicity, poor production in livestock and other associated health problems. These toxic compounds are also responsible for huge economic loss in terms of increased cost in food testing, low-quality agriculture and livestock products, rejection of trade commodities and failure to access international markets. Despite of setting regulatory limits, huge budget allocations in research projects and focused surveillance, aflatoxins are being reported every day in the world especially in developing countries. Pakistan is amongst top milk producing countries of the world but facing aflatoxin contamination problem in raw and processed milk, showing that there exists some deficiencies in decontamination efforts of aflatoxins that are not addressed appropriately and deserve more attention. In addition, when global climate change is brutally affecting food quality and quantity, availability of safe and secure food becomes a major concern. Market indicators have shown that aflatoxins are affecting more than 25% of tradable crops globally and critically responsible for liver cancer and gene mutations. Keeping in view the described scenario, in current study focus has been made on the early detection and decontamination control strategies for aflatoxins. The study describes the morphological and molecular characterization of aflatoxigenic and non aflatoxigenic fungibased on sequencing of ITS regions of fungal genome using universal pair of primers (ITS1 & ITS4). Three Aspergillus species were isolated from feed samples collected from the dairy farms and were identified through phylogenetic analysis to be Aspergillus unguis (GenBank Accession # MH174087, A. niger (GenBank Accession #MH174088)and A. flavus (GenBank Accession # MH179066). The identified aflatoxigenic A. flavus was further analyzed for aflatoxin biosynthetic gene profiling. Two regulatory (aflR, aflS) and five structural (aflQ, aflP, aflD, aflM, aflO) genes were xv targeted using Polymerase Chain Reaction (PCR). The PCR analysis and sequencing of amplicons showed the presence of all targeted genes in the isolated A. flavus, thus its aflatoxigenic nature. The targeted aflatoxigenic gene cluster variation will be helpful in the evolutionary analysis of genetic variation among aflatoxigenic fungi. In the context of “early detection and early control of aflatoxins”, assays were developed and validated for detection of aflatoxin B1 through surface plasmon resonance based immunoassay (biosensor) and ELISA. In case of biosensor based assay, CM5 chip surface was effectively immobilized with AFB1-ova albumin conjugate and used in BIAcore equipment for biospecific interaction analysis. The key analytical features of assay observed were dynamic range, IC20 and IC50 as 3.04-31.9 ng/ml, 3.04 ng/mland 12.7 ng ng/ml, respectively. While in case of developed ELISA, the observed parameters were IC20 as 0.86 ±0.05 (ng g-1), IC50 as 3.62±0.06 ng g-1, CV as 3-14% and average aflatoxin recovery of 99.6%. The assays were based on customized monoclonal antibodies using aflatoxin B1-ova conjugate and presented remarkable analytical characteristics when maize samples spiked with aflatoxins were used as matrix. The potential of developed assays showed that they can be successfully utilized with respect to regulatory requirement of AFB1. Moreover, biological decontamination trials were also performed using viable, heat treated and acid treated lactic acid producing bacterial (LAB) cells. All the LAB cells utilized in current study (Lactobacillus plantarum, L. fermentum, L. paracasei andL. coryniformis) were able to decontaminate the aflatoxin M1from milk samples with variable binding potential when determined using ELISA. Highest binding potential was observed with heat treated cells (92.30-98.77%) as compared to acid treated (68.62-80.63%) and viable LAB cells (56.04-72.64%). The study has potential significance in offering an applicable and point of use approach for the early detection, control and decontamination of aflatoxins.
Gov't Doc #: 23584
URI: http://prr.hec.gov.pk/jspui/handle/123456789/17469
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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