Medical laboratories science

The development of fast, cost-effective, and “eco-friendly” method for the production of silver nanoparticles is an important aspect of nanotechnology today. In this paper, ten fungal strains isolated from marine sediment in Mediterranean Sea (Alexanderia) were screened for their abilities to synthesis silver nanoparticles. Aspergillus terreus MALEX was selected as the most active strain. The silver nanoparticles were characterized by UV–vis spectrophotometry, X-ray diffraction analysis, and Scanning electron microscopy (SEM). UV–visible spectrum of the aqueous medium containing silver ion showed a peak at 420 nm corresponding to the plasmon absorbance of silver nanoparticles. SEM studies showed formation of well-dispersed nanoparticles in the range of 15–29 nm and the shape of nanoparticles was spherical. The biosynthesized
silver nanoparticles exhibited high activities against four pathogenic bacterial strains (Staphylococcus aureus, Klebsiella pneumoniae, E. coli, and Salmonella sp.), four mycotoxigenic fungal strains (Fusarium solani, Alternaria alternata, Aspergillus flavus, A. ochraceus).

Aims: A relative quantification system (RQ-PCR) was used to monitor the
correlations between the activity of the nor-1 (=aflD) gene of Aspergillus flavus
using real-time PCR in relation to phenotypic aflatoxin B1 (AFB1) production
and populations of A. flavus in stored peanuts at three water activity levels (aw,
0Æ95, 0Æ90 and 0Æ85) for 6 weeks.
Methods and Results: Real-time PCR was used to amplify the nor-1 gene
(target gene), and benA56 (b-tubulin gene) used as a control gene. Expression
of three structural genes, nor-1 (=aflD), ver-1 (=aflM), and omtA (=aflP), and
the regulatory gene aflR of the aflatoxin biosynthetic pathway were also
assayed. There were significant differences between nor-1 gene expression at the
three aw levels; higher expression at 0Æ90 aw in weeks 1–3, when compared to
0Æ95. In contrast, in the driest treatment (0Æ85 aw) none or very low nor-1
expression occurred. The populations of A. flavus colony-forming units
(CFUs g)1) increased over time with the highest at 0Æ95 aw. Highest AFB1
production was at 0Æ90 and 0Æ95 aw from weeks 3–6. Aw had a significant effect
on aflR transcription at 0Æ95 aw over the 6-week period, while at 0Æ90 aw, only
in the last 2 weeks.
Conclusions: Correlations between different factors showed that log AFB1 · log
CFUs, log AFB1 · aw, and log CFUs · aw were statistically significant, while log
CFUs · RQ-PCR and RQ-PCR · aw were not. The AflR gene may not have an
important role in the regulation of nor-1 expression in food matrices (e.g.
peanuts).
Significance and Impact of the study: Determination of correlations between
nor-1 expression and aflatoxin production by A. flavus in raw peanuts under
different aw levels could be helpful to predict potential risk of aflatoxin production
during storage of this hygroscopic food product and minimize contamination
with the AFB1.

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B1 (AFB1), and aflatoxin G1 (AFG1) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB1 production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB1 production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB1 production by A. flavus EGP9 and AFG1 production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG1 production by A. parasiticus SSWT 2999. Changes in AFB1 production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between thesestructural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

A wide range of Aspergillus section Flavi strains were isolated from Egyptian peanut samples. 18 of these strains were compared with 2 type strains (A. flavus SRRC G1907, A. parasiticus 2747) for aflatoxin production based on (a) qualitative fluorescence using a coconut cream agar medium (CAM), and (b) aflatoxin production on a conducive Yeast Extract-Sucrose (YES) medium using HPLC. These results were validated by using molecular approaches (the structural genes, aflD (nor-1), aflM (ver-1) and aflP (omt A) and the regulatory gene aflR) to discriminate between aflatoxigenic and non-aflatoxigenic strains of the Aspergillus section Flavi group in vitro and on peanut seeds. Overall, 13/18 strains producing aflatoxin B 1 (AFB 1) and aflatoxin B 2 (AFB 2) in the range 1.27-213.35 μg/g medium were identified. In addition, 5 non-aflatoxin producing strains were found. The expression of these four genes was assessed using PCR (polymerase chain reaction) and RT-PCR (Reverse transcription polymerase chain reaction). PCR showed that all strains contained the four aflatoxin genes examined, regardless of expression profiles. Our results also showed that aflD expression is a reliable marker to discriminate between aflatoxin and non-aflatoxin producers. Interestingly, when an aflatoxin producing strain and three non-aflatoxigenic strains were subsequently grown on peanuts at 0.95 water activity, two of the non-producers were able to initiate aflatoxin biosynthesis. This suggests that growth of strains on the natural food matrix is important for confirming aflatoxigenic production potential.

Recently, methods to analyze aflatoxin M 1 (AFM 1) in milk and dairy products have been developed for both screening purposes (i.e., rapid, economical, and simple methods) and for confirmation by accurate, reproducible, and sensitive quantification. The aim of this study was to evaluate the efficiency of different rapid kits and techniques available on the market by using different analytical methods: thin layer chromatography (TLC), immunoaffinity column, AFM 1 im-munochromatographic strip, and ELISA; some samples were also submitted to HPLC for comparison of results. One hundred thirty-eight samples were collected from rural subsistence and commercial dairy farms in selected areas of Egypt and South Africa and analyzed for the presence of AFM 1. The results obtained by AFM 1 immunochromatographic strip indicated the lowest frequency of occurrence, with a detection incidence of 20.45% in Egyptian samples and 16% in South African samples. Aflatoxin M 1 was detected by ELISA in 65 (73.9%) Egyptian milk samples, with a range of 8.52 to 78.06 ng/L, and in 34 (68%) South African milk samples, with a range of 5 to 120 ng/L. A higher incidence of AFM 1 in Egyptian milk samples was shown by TLC (81.8%) compared with ELISA (73.9%). Samples analyzed by ELISA in South African milk samples demonstrated satisfactory correlation when compared with HPLC coupled with Coring cell (an electrochemi-cal cell for the derivatization of AFM 1). Among the positive samples, 18 of the Egyptian samples (20.45%) positive by ELISA had levels of AFM 1 above the Euro-pean Union (EU) regulatory limit (50 ng/L), whereas 65 samples (73.9%) were above the Egyptian regulatory limit (0 ng/L). Six of the South African samples (12%) tested by ELISA were above the South African (50 ng/L) and EU regulatory limits. The mean concentration of AFM 1 was 25.79 ng/L in Egyptian samples and 17.06 ng/L by ELISA and 39 ng/L by HPLC in South African samples. These contamination levels would not represent a serious public health hazard according to EU legislation.