Journal of microbiological methods最新文献

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.

Short-chain organic acids (SCOAs) are the intermediates in the anaerobic fermentation process, and can be used in food, textile, and pharmaceutical industries to produce different end use products. SCOAs can be separated, purified, and concentrated by different processes, such as distillation, extraction or membrane-based systems. SCOAs production adds more profitable possibilities to an acidic fermentation process by integration these marketable acids as highly concentrated mixtures with other refinery processes. The present study investigated two approaches for recovering of SCOAs: i) the production of clarified SCOAs liquid by microfiltration (MF) and then performing their concentration by reverse osmosis (RO) and ii) the recovery and concentration by the so-called integrated neutralization and acidified reaction method. The results of MF showed that some SCOAs were retained in the retentate together with the solids. However, in the following RO treatment, SCOAs could be successfully concentrated with a yield retention of over 90 % from the SCOAs liquid. In the latter method, a color-free SCOAs liquid was obtained with an increase in the total SCOAs concentration from 23 g/L to 146 g/L.

Mycoplasma genitalium (MG) is an important sexually transmitted pathogen that can cause urethritis in males and pelvic inflammatory disease in females. Due to its complex growth requirements and lengthy incubation times, culturing MG in clinical laboratories is impractical. Here we describe a rapid and visual assay combining recombinase polymerase amplification (RPA) with lateral flow (LF) strips to detect MG (MG-RPA-LF). The limit of detection (LoD) of this method was 33.6 genome equivalents (GE) per reaction, using a dilution series of purified genomic DNA. Clinical performance was evaluated by testing 100 urogenital swabs. Compared to the Simultaneous Amplification and Testing assay, our MG-RPA-LF assay showed a sensitivity of 94 % (95 % CI, 82 %–98 %) and a specificity of 100 % (95 % CI, 91 %–100 %). The overall concordance between the two methods was 97 % (95 % CI, 91 %–99 %) with a κ coefficient of 0.94 (P < 0.001). Without cumbersome and expensive instruments, this method is anticipated to be a promising alternative to diagnose MG infection, especially in resource-poor settings.

The use of detergents when culturing Mycobacterium tuberculosis (M. tuberculosis) are essential to prevent clumping. However, these detergents may influence research outcomes by impacting bacterial morphology and metabolism. This study aimed to assess the metabolome of a M. tuberculosis H37Rv strain cultured with Tyloxapol (H37Rv^Tyloxapol), compared to a control group of H37Rv strain cultured without detergent (H37Rv^Control) to evaluate Tyloxapol's suitability for metabolomic studies. Distinct metabolic alterations were observed in H37Rv^Tyloxapol compared to H37Rv^Control, primarily associated with fatty acid, sugar and pentose phosphate metabolic pathways. These changes are associated with the surface stress exerted by Tyloxapol on the bacteria, prompting an adaptation of M. tuberculosis metabolism to that usually observed in stress environments. Nevertheless, the effect of Tyloxapol is less pronounced than that of a previous investigation using Tween 80, indicating its potential as the more favourable choice for culturing M. tuberculosis for metabolomic analysis, with due consideration to dosage and result interpretation.

HSD-IO01, a new pure strain of I. obliquus, was isolated and purified from the sclerotium of I. obliquus of Daxing'an Mountains. Physical radiation-assisted liquid fermentation technology was explored to increase the triterpenoids yield of HSD-IO01. In the 100 mL optimized liquid fermentation system, the hypha dry weight of HSD-IO01 was 1.7734 g, and the triterpenoids yield was 43.43 mg. Yields of triterpenoids increased after induction with ultrasound, microwave, or UV light, respectively. Among them, ultrasonic treatment had the most remarkable induction effect. The yield of triterpenoids would be increased to 68.35 mg (57.38 %) when the HSD-IO01 was treated by 100 W ultrasonic for 45 min. Establishing ultrasonic-assisted liquid fermentation technology could further promote the detailed development and comprehensive utilization of I. obliquus resources.

There is interest in studying microbes that colonize maize silks (style tissue, critical for reproduction) including the fungal pathogen Fusarium graminearum (Fg) and its interactions with the microbiome and biocontrol agents. In planta imaging of these interactions on living silks using confocal fluorescence microscopy would provide key insights. However, newly discovered microbes have unknown effects on human health, and there are regulatory requirements to prevent the release of fluorescently tagged microbes into the environment. Therefore, the microbe infection, colonization, and interaction stages on silks prior to microscopy must be contained. At the same time, silk viability must be maintained and experiments conducted that are biologically relevant (e.g. silks should remain attached to the cob), yet the silk tissue must be accessible to the researcher (i.e. not within husk leaves) and allow for multiple replicates. Here we present methods that meet these five contrasting criteria. We tested these methods using Fg and four silk-derived bacterial endophytes. The endophytes were previously known to have anti-Fg activity in vitro, but in planta observations were lacking. In Method 1, a portion of the tip of a cob was dissected, and silks remained attached to the cob in a Petri dish. The cob was placed on a water agar disc to maintain hydration. DsRed-tagged bacteria and GFP-tagged Fg were inoculated onto the silks and incubated, allowing the two microbes to grow towards one another before staining with propidium iodide for confocal microscopy. A variation of the protocol was presented in Method 2, where detached silk segments were placed directly on water agar where they were inoculated with bacteria and Fg to promote dense colonization, and to allow for many replicates and interventions such as silk wounding. The bacterial endophytes were successfully observed colonizing Fg hyphae, silk trichomes, and entering silks via cut ends and wounds. These protocols can be used to study other silk-associated microbes including several globally important fungal pathogens that enter maize grain through silks.

Purpose

The opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method.

Methods

Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis.

Results

This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 10² copies/μL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %.

Conclusion

In this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.