Search Results (9)

Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 10²–10⁸ CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA–metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms. Full article

Most previously studies had considered that plant fungal disease spread widely and quickly by airborne fungi spore. However, little is known about the release dynamics, aerodynamic diameter, and pathogenicity threshold of fungi spore in air of the greenhouse environment. Grape gray mold is caused by Botrytis cinerea; the disease spreads in greenhouses by spores in the air and the spore attaches to the leaf and infects plant through the orifice. In this study, 120 μmol/L propidium monoazide (PMA) were suitable for treatment and quantitation viable spore by quantitative real-time PCR, with a limit detection of 8 spores/mL in spore suspension. In total, 93 strains of B. cinerea with high pathogenicity were isolated and identified from the air samples of grapevines greenhouses by a portable sampler. The particle size of B. cinerea aerosol ranged predominately from 0.65–3.3 μm, accounting for 71.77% of the total amount. The B. cinerea spore aerosols were infective to healthy grape plants, with the lowest concentration that could cause disease being 42 spores/m³. Botrytis cinerea spores collected form six greenhouse in Shandong Province were quantified by PMA-qPCR, with a higher concentration (1182.89 spores/m³) in May and June and a lower concentration in July and August (6.30 spores/m³). This study suggested that spore dispersal in aerosol is an important route for the epidemiology of plant fungal disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant diseases. Full article

The current study investigated the effects of heat treatment (85 °C or 100 °C for 5–20 min) on human norovirus (HuNoV) GII.4’s capsid stability in fresh oysters. In addition, propidium monoazide (PMA) was used in viral samples to distinguish infectious viruses and evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Further, we explored the effect of the heat treatment on oyster quality (Hunter color and hardness). The titer of HuNoV for oysters significantly (p < 0.05) decreased to 0.39–1.32 and 0.93–2.27 log₁₀ copy number/μL in the non-PMA and PMA-treated groups, respectively, after heat treatment. HuNoV in oysters not treated with PMA showed a decrease of <1.5 − log₁₀, whereas in PMA-treated oysters, a decrease of >1 − log₁₀ was observed after treatment at 85 °C for 10 min. Treatments for both 15 min and 20 min at 100 °C showed a >99% log₁₀ reduction using PMA/RT-qPCR. In the Hunter color, an increase in heat temperature and duration was associated with a significant decrease in ‘L’ (brightness+, darkness−) and an increase in ‘a’ (redness+, greenness−) and ‘b’ (yellowness+, blueness−) (p < 0.05). Our findings confirmed that the hardness of oyster meat significantly increased with increasing temperature and time (p < 0.05). This study demonstrated that PMA/RT-qPCR was effective in distinguishing HuNoV viability in heat-treated oysters. The optimal heat treatment for oysters was 10 min at 85 °C and 5 min at 100 °C. Full article

The authenticity of probiotic products and fermented foods and beverages that have the status of protected designation of origin (PDO) or geographical indication (PGI) can be assessed via numerous methods. DNA-based technologies have emerged in recent decades as valuable tools to achieve food authentication, and advanced DNA-based methods and platforms are being developed. The present review focuses on the recent and advanced DNA-based techniques for the authentication of probiotic, PDO and PGI fermented foods and beverages. Moreover, the most promising DNA-based detection tools are presented. Strain- and species-specific DNA-based markers of microorganisms used as starter cultures or (probiotic) adjuncts for the production of probiotic and fermented food and beverages have been exploited for valuable authentication in several detection methods. Among the available technologies, propidium monoazide (PMA) real-time polymerase chain reaction (PCR)-based technologies allow for the on-time quantitative detection of viable microbes. DNA-based lab-on-a-chips are promising devices that can be used for the on-site and on-time quantitative detection of microorganisms. PCR-DGGE and metagenomics, even combined with the use of PMA, are valuable tools allowing for the fingerprinting of the microbial communities, which characterize PDO and PGI fermented foods and beverages, and they are necessary for authentication besides permitting the detection of extra or mislabeled species in probiotic products. These methods, in relation to the authentication of probiotic foods and beverages, need to be used in combination with PMA, culturomics or flow cytometry to allow for the enumeration of viable microorganisms. Full article

Human norovirus (HuNoV) is a common cause of outbreaks linked to food. In this study, the effectiveness of a non-thermal method known as high-pressure processing (HPP) on the viable reduction of an HuNoV GII.4 strain on raw crabs was evaluated at three different pressures (200, 400, and 600 MPa). HuNoV viability in raw crabs was investigated by using propidium monoazide/sarkosyl (PMA) as a nucleic acid intercalating dye prior to performing a real-time reverse transcription-polymerase chain reaction (RT-qPCR). The effect of the HPP exposure on pH, sensory, and Hunter colors were also assessed. HuNoV was reduced in raw crabs compared with control to HPP (0.15–1.91 log) in non-PMA and (0.67–2.23 log) in PMA. HuNoV genomic titer reduction was <2 log copy number/µL) when HPP was treated for 5 min without PMA pretreatment, but it was reduced to >2 log copy number/µL after PMA. The pH and Hunter colors of the untreated and HPP-treated raw crabs were significantly different (p < 0.05), but sensory attributes were not significant. The findings indicate that PMA/RT-qPCR could be used to detect HuNoV infectivity without altering the quality of raw crabs after a 5 min treatment with HPP. Therefore, HuNoV GII.4 could be reduced up to 2.23 log in food at a commercially acceptable pressure duration of 600 MPa for 5 min. Full article

Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is an important quarantine virus of cucurbit crops. Seedborne transmission is one of the principal modes for CGMMV spread, and effective early detection is helpful to prevent the occurrence of the disease. Quantitative real-time reverse-transcription PCR (RT-qPCR) is a sensitive and rapid method for detecting CGMMV nucleic acids, but it cannot distinguish between infectious and noninfectious viruses. In the present work, a propidium monoazide (PMA) assisted RT-qPCR method (PMA-RT-qPCR) was developed to rapidly distinguish infectious and inactive CGMMV. PMA is a photoactive dye that can selectively react with viral RNA released or inside inactive CGMMV virions but not viral RNA inside active virions. The formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be amplified. The primer pair cp3-1F/cp3-1R was designed based on the coat protein (cp) gene for specific amplification of CGMMV RNA by RT-qPCR. The detection limit of the RT-qPCR assay was 1.57 × 10² copies·μL⁻¹. PMA at 120 μmol·L⁻¹ was suitable for the selective quantification of infectious CGMMV virions. Under optimal conditions, RT-qPCR detection of heat-inactivated CGMMV resulted in Ct value differences larger than 16 between PMA-treated and non-PMA-treated groups, while Ct differences less than 0.23 were observed in the detection of infectious CGMMV. For naturally contaminated watermelon leaf, fruit and seedlot samples, infectious CGMMV were quantified in 13 out of the 22 samples, with infestation levels of 10²~10⁵ copies·g⁻¹. Application of this assay enabled the selective detection of infectious CGMMV and facilitated the monitoring of the viral pathogen in watermelon seeds and tissues, which could be useful for avoiding the potential risks of primary inoculum sources. Full article

Fusarium is a soil-borne pathogen that causes root rot disease in cucumber. To date, quantitative real-time PCR (qPCR) is a common tool to detect the content of Fusarium in soil. However, qPCR cannot distinguish between viable and nonviable cells. The aim of this study was to develop a detection technique to pretreat tissue fluid with propidium monoazide (PMA) followed by extract DNA, and then to quantify viable Fusarium cells in contaminated soil. In this work, the specific primer pair F8-1/F8-2 was designed based on the translation elongation factor (EF) gene and a PMA-qPCR assay was established to amplify and quantify soils of viable Fusarium cells. The PMA pretreatment test was optimized, which indicated that the optimal PMA concentration and light exposure time were 50 mmol L⁻¹ and 15 min, respectively. The lowest limit of viable cells in suspension detected and soil by PMA-qPCR were 82 spore mL⁻¹ and 91.24 spore g⁻¹, respectively. For naturally contaminated soil, viable Fusarium cells were detected in eight of the 18 samples, and the Fusarium amount ranged from 10⁴ to 10⁶ spore g⁻¹. In conclusion, the PMA-qPCR method has the characteristics of high sensitivity, efficiency, and time saving, which could support nursery plants to avoid Fusarium infection and agro-industry losses. Full article

The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLight^TM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLight^TM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared Full article

This study investigates the effects of dielectric barrier discharge (DBD) plasma treatment (1.1 kV, 43 kHz, N₂ 1.5 L/min, 10~60 min) on human norovirus (HuNoV) GII.4 infectivity in fresh oysters. HuNoV viability in oysters was assessed by using propidium monoazide (PMA) as a nucleic acid intercalating dye before performing a real-time reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Additionally, the impact of the DBD plasma treatment on pH and Hunter colors was assessed. When DBD plasma was treated for 60 min, the HuNoV genomic titer reduction without PMA pretreatment was negligible (<1 log copy number/µL), whereas when PMA treatment was used, HuNoV titer was reduced to >1 log copy number/µL in just 30 min. D₁ and D₂-value of HuNoV infectivity were calculated as 36.5 and 73.0 min of the DBD plasma treatment, respectively, using the first-order kinetics model (R² = 0.98). The pH and Hunter colors were not significantly different (p > 0.05) between the untreated and DBD-plasma-treated oysters. The results suggest that PMA/RT-qPCR could help distinguish HuNoV infectivity without negatively affecting oyster quality following >30 min treatment with DBD plasma. Moreover, the inactivation kinetics of nonthermal DBD plasma against HuNoV in fresh oysters might provide basic information for oyster processing and distribution. Full article