Numerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly. In this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1–26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis. The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin, which deserves particular attention for its widespread contamination of a variety of food and feed. Aspergillus ochraceus, Aspergillus carbonarius, and Penicillium nordicum are an important source of OTA in three different kinds of food commodities, including cereals, grape and dried fruit products, and dry-cured meat products. Deeper knowledge of OTA production and mycelium growth related to the high-sugar or NaCl-rich environments was gained in this manuscript. A. ochraceus and P. nordicum were likely to have greater growth rates in medium supplied with certain concentrations of NaCl (0–80 g/L), and the colony diameter was the largest at the salt content of 40 g/L. P. nordicum was more suitable to grow in NaCl-riched medium, the OTA production was increased to 316 ppb from 77 ppb when 20 g/L NaCl was added. The capability of OTA production was inhibited when salt content was 40 g/L and 60 g/L in A. ochraceus and P. nordicum, respectively. As the glucose content increased to 250 g/L, the capacity of mycelium growth and sporulation was increased significantly in A. ochraceus and A. carbonarius. A. carbonarius was more suitable to grow in high-sugar grape products. OTA production was significantly promoted with an added 100 g/L glucose in A. carbonarius. OTA production was inhibited when glucose content was 150 g/L and in 200 g/L in A. ochraceus and A. carbonarius, respectively. NaCl and glucose have an effect on fungal growth and OTA production, and the activation of biosynthetic genes of OtaA. These results would allow designing new strategies to prevent OTA accumulation on sugar or NaCl-riched foodstuffs and achieve the objective to manufacture cereals, dried vine fruits and dry-cured ham, free of OTA.

Aspergillus westerdijkiae, known as the major ochratoxin A (OTA) producer, usually occurs on agricultural crops, fruits, and dry-cured meats. Microorganisms produce OTA to adapt to the high osmotic pressure environment that is generated during food processing and storage. To investigate the relationship between OTA biosynthesis and the high osmolarity glycerol (HOG) pathway, the transcription factor AwHog1 gene in A. westerdijkiae was functionally characterised by means of a loss-of-function mutant. Our findings demonstrated that the growth and OTA production of a mutant lacking AwHog1 decreased significantly and was more sensitive to high osmotic media. The ΔAwHog1 mutant displayed a lower growth rate and a 73.16% reduction in OTA production in the wheat medium compared to the wild type. After three days of culture, the growth rate of the ΔAwHog1 mutant in medium with 60 g/L NaCl and 150 g/L glucose was slowed down 19.57% and 13.21%, respectively. Additionally, the expression of OTA biosynthesis genes was significantly reduced by the deletion of the AwHog1 gene. The infection ability of the ΔAwHog1 mutant was decreased, and the scab diameter of the pear was 6% smaller than that of the wild type. These data revealed that transcription factor AwHog1 plays a key role in the osmotic response, growth, OTA production, and pathogenicity in A. westerdijkiae.

Dry-cured meat products are worldwide food with high-salt content, and filamentous fungi are beneficial to the maturation process. However, some salt-tolerant strains of Aspergillus and Penicillium produce ochratoxin A (OTA) on these products and thus threaten food safety. In our study, proteomic analysis was performed to reveal the mechanism of adaptability to high-salt environment by Aspergillus ochraceus. Twenty g/L and 70 g/L NaCl substrates were used to provide medium- and high-NaCl content environments, respectively. The NaCl addition could induce fungal growth, but only 20 g/L NaCl addition could induce spore production while 70 g/L repressed it. Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 237 and 251 were differentially expressed with 20 g/L and 70 g/L NaCl addition, respectively. Potential factors affecting fungal growth and development were identified by GO and KEGG analyses of biological process, cellular component, and molecular function terms. The results revealed that ergosterol synthesis pathway was significantly upregulated with 20 g/L and 70 g/L NaCl addition. However, fungal growth and development including OTA production were complex processes associated with many factors including nutrient uptake, cell membrane integrity, cell cycle, energy metabolism, intracellular redox homeostasis, protein synthesis and processing, autophagy, and secondary metabolism. Reactive oxygen species may be an important window to understand the mechanism that medium-salt content was conducive to intracellular signal transduction while high-salt content caused oxidative stress. The findings would help to improve the processes and storage conditions of dry-cured meat products.