Journal of microbiological methods最新文献

Background

Aim

Methods

Results

Conclusions

Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium Ecytonucleospora hepatopenaei (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against Cryptosporidium parvum, a coccidian parasite that infects calves (Bos taurus), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (Penaeus vannamei). EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of C. parvum has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease.

A rapid and cost-effective method for detecting bacterial cells from surfaces is critical to food safety, clinical hygiene, and pharmacy quality. Herein, we established an optical detection method based on a gold chip coating with 3-mercaptophenylboronic acid (3-MPBA) to capture bacterial cells, which allows for the detection and quantification of bacterial cells with a standard light microscope under low-magnification (10×) objective lens. Then, integrate the developed optical detection method with swab sampling to detect bacterial cells loading on stainless-steel surfaces. Using Salmonella enterica (SE1045) and Escherichia coli (E. coli OP50) as model bacterial cells, we achieved a capture efficiency of up to 76.0 ± 2.0 % for SE1045 cells and 81.1 ± 3.3 % for E. coli OP50 cells at 10³ CFU/mL upon the optimized conditions, which slightly decreased with the increasing bacterial concentrations. Our assay showed good linear relationships between the concentrations of bacterial cells with the cell counting in images in the range of 10³ -10⁷ CFU/mL for SE1045, and 10³ -10⁸ CFU/mL for E. coli OP50 cells. The limit of detection (LOD) was 10³ CFU/mL for both SE1045 and E. coli OP50 cells. A further increase in sensitivity in detecting E. coli OP50 cells was achieved through a heat treatment, enabling the LOD to be reduced as low as 10² CFU/mL. Furthermore, a preliminary application succeeded in assessing bacterial contamination on stainless-steel surfaces following integration with the approximately 40 % recovery rate, suggesting prospects for evaluating the bacteria from surfaces. The entire process was completed within around 2 h, costing merely a few dollars per sample. Considering the low cost of standard light microscopes, our method holds significant potential for practical industrial applications in bacterial contamination control on surfaces, especially in low-resource settings.

We evaluated the analytical performance of three commercial molecular assays for rapid detection of Clostridioides difficile toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 C. difficile and Cepheid Xpert® C. difficile BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 C. difficile assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic C. difficile.

Nested PCR is a useful tool for identifying low-abundance target sequences of pathogens and avoiding false negatives. However, it carries an increased risk of cross-contamination, especially with its positive control. Here, we propose using customized synthetic oligonucleotides to detect false positives due to cross-contamination.

As an extremophile resource, functional Haloarchaea strains are extremely time-consuming to screen. Here, taking the screening of low-salt-tolerant strains as an example, based on the qPCR assays that shortened time by 4–7 times and achieved 100 % accuracy, a universal strategy for rapid and accurate screening of functional Haloarchaea strains was established.