scholarly journals Molecular Identification of Aflatoxigenic Fungi in Some Foods from Selected Markets in Lagos

2020 ◽  
pp. 42-50
Author(s):  
Oluwatosin Bidemi Ajiboye ◽  
Wahab Oluwanisola Okunowo ◽  
Emmanuel Gboyega Ajiboye ◽  
Abiola Olajumoke Oyedeji
Keyword(s):  
Molecular Identification ◽  
Genomic Dna ◽  
Regulatory Gene ◽  
Pcr Amplification ◽  
High Specificity ◽  
Food Samples ◽  
Local Market ◽  
Nested Polymerase Chain Reaction ◽  
Aflatoxigenic Fungi ◽  
Food Commodities

Aflatoxigenic fungi are species of fungi that produce aflatoxins in food commodities. This study was aimed at screening different food samples in our local market for aflatoxigenic fungi using the aflatoxin regulatory gene (aflR gene). Six food samples (wheat, cowpea, rice, maize, melon and groundnut), were sourced from three different markets in Lagos metropolis (Mushin, Oyingbo and Mile 12). Fungi were isolated from these food samples and identified morphologically and microscopically. The genomic DNA was obtained using DNA isolation kits. The aflR gene was amplified from genomic DNA, nested, subjected to agarose gel electrophoresis and gel imaging. The Internal Transcribed Spacer (ITS) was also amplified from the genomic DNA for molecular identification of the organisms. The results showed that Aspergillus flavus were isolated from all the food samples from the three markets, while Aspergillus niger was present in rice, melon and wheat from Mile 12 market, maize and groundnut from Mushin market, rice and cowpea from Oyingbo market. A. flavus and A.niger were isolated from all the food samples when similar food samples from different market were mixed together. Only A. flavus amplicon from the nested polymerase chain reaction (PCR) showed approximately 400bp DNA fragment on the gel. This study has shown that PCR amplification of aflR gene has high specificity for detection of aflatoxigenic fungi in food samples thus, may be employed in screening food samples for contamination by aflatoxigenic fungi.

A neutral red desiccated coconut agar for rapid detection of aflatoxigenic fungi and visual determination of aflatoxins

2011 ◽  
Vol 4 (2) ◽  
pp. 147-155 ◽  
Author(s):  
O. Atanda ◽  
M. Ogunrinu ◽  
F. Olorunfemi
Keyword(s):  
Fish Meal ◽  
Palm Kernel ◽  
Neutral Red ◽  
Food Samples ◽  
White Background ◽  
Phenol Red ◽  
Aflatoxigenic Fungi ◽  
Aflatoxin Concentration ◽  
Food Commodities

Desiccated coconut agar is the conventional medium used for the detection of aflatoxigenic fungi and direct visual determination of aflatoxins. In this study, an improved medium was developed by the incorporation of 0.2% (v/v) neutral red dye into desiccated coconut agar. The medium was formulated by a 2×3 factorial design of neutral red and phenol red stains at three concentration levels. The formulated medium was evaluated for performance by screening for the minimal time required by each Aspergillus species to produce pigments and fluorescence of agar. The medium was also employed for detection of aflatoxigenic fungi and direct visual determination of aflatoxins in foods and fish-meal. The neutral red desiccated coconut agar (NRDCA) as compared to the conventional desiccated coconut agar (DCA) had a light pink background as opposed to the white background of the DCA which often interferes with the visibility of fluorescence. The time of pigmentation and fluorescence production on NRDCA was 28 and 38 h respectively as compared with 33 and 44 h of DCA and 41 and 48 h of palm kernel agar (PKA: an alternative culture medium for cultivation of aflatoxigenic fungi with a reddish pink background). Furthermore, aflatoxigenic moulds were detected in all food commodities and fish-meal after 60 hours of incubation. The highest percentage of aflatoxigenic moulds (62.5%) was detected in yam flour with NRDCA while the lowest percentage (4.46%) was detected with PKA on rice. In addition, aflatoxins were produced in high amounts in food commodities in which aflatoxigenic moulds were detected and there was a significant positive correlation (r=0.4, P<0.05) between the isolates and aflatoxin concentration of the food samples. Rice (a major staple food for Nigerians) had the highest total aflatoxin concentration of 140, 220 and 205 µg/kg on DCA, NRDCA and PKA respectively, while ‘gari’ had the least concentration of 45, 50 and 40 µg/kg. These values were far above the NAFDAC recommended level of 10 µg/kg for unprocessed foods in Nigeria and therefore a source of concern. In addition the study also reveals that Aspergillus nomius can produce aflatoxins B1 in copious amounts on NRDCA, contrary to previous reports of its production in minute quantities on laboratory media. The benefit of this study lies in the rapid analysis and simplified technique for the detection of aflatoxigenic fungi and visual determination of aflatoxins.

scholarly journals Cloning and Characterization of a Chromosomal Class C β-Lactamase and Its Regulatory Gene in Laribacter hongkongensis

2005 ◽  
Vol 49 (5) ◽  
pp. 1957-1964 ◽  
Author(s):  
Susanna K. P. Lau ◽  
Pak-leung Ho ◽  
Maria W. S. Li ◽  
Hoi-wah Tsoi ◽  
Raymond W. H. Yung ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Southern Hybridization ◽  
Regulatory Gene ◽  
Kinetic Properties ◽  
Restriction Fragments ◽  
Regulatory Systems ◽  
Laribacter Hongkongensis ◽  
Consensus Motifs ◽  
Class C

ABSTRACT Laribacter hongkongensis, a newly discovered bacterium recently shown to be associated with community-acquired gastroenteritis, is generally resistant to most β-lactams except the carbapenems. We describe the cloning and characterization of a novel chromosomal class C β-lactamase and its regulatory gene in L. hongkongensis. Two genes, ampC and ampR, were cloned by inserting restriction fragments of genomic DNA from L. hongkongensis strain HLHK5 into pBK-CMV to give the recombinant plasmid pBK-LHK-5. The ampR and ampC genes and their promoters were divergently oriented, with the ampR gene immediately upstream of the ampC gene and an intercistronic Lys-R motif, typical of inducible ampC-ampR regulatory systems. The deduced amino acid sequence of the cloned AmpC β-lactamase (pI 8.1) contained consensus motifs characteristic of class C β-lactamases but had identities no greater than 46% to known class C β-lactamases. The kinetic properties of this AmpC were also compatible with those of a class C β-lactamase. PCR of 20 clinical isolates of L. hongkongensis, including HLHK5, showed the presence of both ampC and ampR genes in all isolates. Southern hybridization suggested that the ampC gene of HLHK5 was chromosomally encoded. Subcloning experiments showed that the expression of the ampC gene of HLHK5 was regulated by its ampR gene, which acts as a repressor. The β-lactamase characterized from strain HLHK5 was named LHK-5 (gene, bla LHK-5) and represents the first example of AmpC β-lactamase in the β subdivision of proteobacteria.

scholarly journals Prevalence of UL97 gene mutations and polymorphisms in cytomegalovirus infection in the colon associated with or without ulcerative colitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Satoshi Tamura ◽  
Satoshi Osawa ◽  
Natsuki Ishida ◽  
Takahiro Miyazu ◽  
Shinya Tani ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Kinase Domain ◽  
Resistance Mutations ◽  
Cmv Infection ◽  
Nested Polymerase Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ul97 Gene

AbstractCytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. In comparison with AD169 reference strain, natural polymorphisms were frequently detected in codons N68D (100%), I244V (100%), and D605E (86.4%). Seven polymorphisms were detected infrequently (< 10%) outside the kinase domain. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of polymorphisms in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. Consistent with previous reports, D605E polymorphism may be used as a genetic marker for CMV in East Asian countries.

scholarly journals Single and Double Infections with Wolbachia in the Parasitic Wasp Nasonia vitripennis Effects on Compatibility

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 961-972 ◽  
Author(s):  
Marie-Jeanne Perrot-Minnot ◽  
Li Rong Guo ◽  
John H Werren
Keyword(s):  
Genomic Dna ◽  
Rapid Development ◽  
Parasitic Wasp ◽  
Pcr Amplification ◽  
Bacterial Strains ◽  
Nasonia Vitripennis ◽  
Larval Diapause ◽  
Wide Range ◽  
Reproductive Incompatibility ◽  
Infected Line

Abstract Wolbachia are cytoplasmically inherited bacteria responsible for reproductive incompatibility in a wide range of insects. There has been little exploration, however, of within species Wolbachia polymorphisms and their effects on compatibility. Here we show that some strains of the parasitic wasp Nasonia vitripennis are infected with two distinct bacterial strains (A and B) whereas others are singly infected (A or B). Double and single infections are confirmed by both PCR amplification and Southern analysis of genomic DNA. Furthermore, it is shown that prolonged larval diapause (the overwintering stage of the wasp) of a double-infected strain can lead to stochastic loss of one or both bacterial strains. After diapause of a double-infected line, sublines were produced with AB, A only, B only or no Wolbachia. A and B sublines are bidirectionally incompatible, whereas males from AB lines are unidirectionally incompatible with females of A and B sublines. Results therefore show rapid development of bidirectional incompatibility within a species due to segregation of associated symbiotic bacteria.

scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

scholarly journals Morphological and molecular identification of phytophthora species from maple trees in Serbia

Genetika ◽  
2014 ◽  
Vol 46 (2) ◽  
pp. 353-368 ◽  
Author(s):  
Ivan Milenkovic ◽  
Justyna Nowakowska ◽  
Tomasz Oszako ◽  
Katarina Mladenovic ◽  
Aleksandar Lucic ◽  
...  
Keyword(s):  
Molecular Identification ◽  
Genomic Dna ◽  
Its Region ◽  
Phytophthora Species ◽  
Morphological Identification ◽  
First Report ◽  
Phytophthora Spp ◽  
Isolated Species

The paper presents the results of the study performed with aims to determine the presence and diversity of Phytophthora species on maple trees in Serbia. Due to high aggressiveness and their multicyclic nature, presence of these pathogens is posing significant threat to forestry and biodiversity. In total, 29 samples of water, soil and tissues were taken from 10 different localities, and six different maple hosts were tested. After the isolation tests, 17 samples from five different maple hosts were positive for the presence of Phytophthora spp., and 31 isolates were obtained. After the detailed morphological and physiological classification, four distinct groups of isolates were separated. DNA was extracted from selected representative isolates and molecular identification with sequencing of ITS region was performed. Used ITS4 and ITS6 primers successfully amplified the genomic DNA of chosen isolates and morphological identification of obtained isolates was confirmed after the sequencing. Four different Phytophthora species were detected, including P. cactorum, P. gonapodyides, P. plurivora and P. lacustris. The most common isolated species was homothallic, and with very variable and semipapillate sporangia, P. plurivora with 22 obtained isolates. This is the first report of P. plurivora and P. gonapodyides on A. campestre, P. plurivora and P. lacustris on Acer heldreichii and first report of P. lacustris on A. pseudoplatanus and A. tataricum in Serbia.

Identification of developmental regulatory genes in Aspergillus nidulans by overexpression.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 537-547 ◽  
Author(s):  
J F Marhoul ◽  
T H Adams
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Nidulans ◽  
Genomic Dna ◽  
Regulatory Gene ◽  
Regulatory Genes ◽  
Dna Fragments ◽  
Expression Library ◽  
Phenotypic Changes ◽  
Inhibition Of Growth ◽  
Induction Sequence

Abstract Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [alcA(p)] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA(p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA(p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA(p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA(p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA(p)::brlA induction. Sequence analyses of the DNA fragments under alcA(p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA(p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.

scholarly journals Prenatal diagnosis of pyruvate kinase deficiency

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2354-2356 ◽  
Author(s):  
L Baronciani ◽  
E Beutler
Keyword(s):  
Pyruvate Kinase ◽  
Genomic Dna ◽  
Direct Method ◽  
Pcr Amplification ◽  
Prenatal Testing ◽  
Restriction Endonuclease Analysis ◽  
Diagnostic Methods ◽  
Pyruvate Kinase Deficiency ◽  
A Direct Method ◽  
Endonuclease Analysis

Abstract Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.

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