Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk.
ABSTRACT: Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS-PMA-qPCR, PMA-qPCR and qPCR when a final concentration of 10 mg/ml (R 2 = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA-qPCR. Moreover, the SDS-PMA-qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 103 cfu/ml. Therefore, the SDS-PMA-qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk.
Project description:BACKGROUND:Staphylococcus aureus is an important human pathogen causing a variety of life-threatening diseases. Rapid and accurate detection of Staphylococcus aureus is a necessity for prevention of outbreaks caused by this pathogen. PCR is a useful tool for rapid detection of foodborne pathogens, however, its inability to differentiate DNA from dead cells and live cells in amplification severely limits its application in pathogen detection. The aim of this study was to develop an improved assay was developed by incorporating the sample treatments with a surfactant and propidium monoazide (PMA) in qPCR for detection of viable S. aureus cells. RESULTS:The cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. Triton x-100 was coupled with PMA for sample treatments for detection of viable S. aureus cells in artificially contaminated milk. The qPCR results indicated that the assay reached high an amplification efficiency of 98.44% and the live S. aureus cells were accurately detected from the triton-treated spiked milk samples by the PMA-qPCR assay. CONCLUSIONS:The qPCR assay combined with treatments of PMA and surfactants offers a sensitive and accurate means for detection of viable S. aureus cells. Cell toxic effect testing with the two surfactants showed that the viability of S. aureus was virtually not affected by the treatment with 0.5% triton x-100 or 0.025% sarkosyl. The information on sample treatment with surfactants to improve the dead cell DNA removal efficiency in qPCR by increasing PMA's permeability to dead cells can be used for other pathogens, especially for Gram-positive bacteria.
Project description:Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 10(8) CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 10(3) to 6.1 × 10(4) CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (T(T)) values and viable Salmonella cell numbers was high (R(2) = 0.949 to 0.993), with a quantification range (10(2) to 10(5) CFU/reaction in pure culture and 10(4) to 10(7) CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.
Project description:Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable Escherichia coli O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 ?M PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 102colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (107 CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of E. coli O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC E. coli O157:H7 in agricultural soil.
Project description:We developed a PCR-based method to detect and quantify viable Bifidobacterium bifidum BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 10(10) to 10(6) cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4',6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 10(5.3) to 10(10.3) cells/g feces (wet weight) (r > 0.99, P < 0.001). After 12 healthy subjects ingested 10(10.3) to 10(11.0) CFU of BF-1 in a fermented milk product daily for 28 days, 10(4.5 ± 1.5) (mean ± standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 10(6.2 ± 0.4) viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 10(7.6 ± 0.7) BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (P < 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces.
Project description:We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4?×?103, 4.8?×?103, and 7.2?×?103 ?J/cm2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8?×?103 ?J/cm2 in 3 min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 100 CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4 h.
Project description:Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (108 CFU mL-1) was induced by 50 ?M copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 ?M for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 103 to 107 CFU mL-1. The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.
Project description:Campylobacter can enter a viable but non-culturable (VBNC) state to evade various stresses, and this state is undetectable using traditional microbiological culturing techniques. These VBNC bacterial cells retain metabolism and demonstrate pathogenic potential due to their ability to resuscitate under favorable conditions. Rapid and accurate determination of VBNC Campylobacter is critical to further understand the induction and resuscitation of the dormancy state of this microbe in the agri-food system. Here, we integrated propidium monoazide (PMA) with real-time polymerase chain reaction (qPCR) targeting the rpoB gene to detect and quantify Campylobacter jejuni in the VBNC state. First, we optimized the concentration of PMA (20 ?M) that could significantly inhibit the amplification of dead cells by qPCR with no significant interference on the amplification of viable cell DNA. PMA-qPCR was highly specific to C. jejuni with a limit of detection (LOD) of 2.43 log CFU/ml in pure bacterial culture. A standard curve for C. jejuni cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 3.43 to 8.43 log CFU/ml. Induction of C. jejuni into the VBNC state by osmotic stress (i.e., 7% NaCl) was rapid (<48 h) and effective (>10% population). The LOD of PMA-qPCR for VBNC C. jejuni exogenously applied to chicken breasts was 3.12 log CFU/g. In conclusion, PMA-qPCR is a rapid, specific, and sensitive method for the detection and quantification of VBNC C. jejuni in poultry products. This technique can give insight into the prevalence of VBNC Campylobacter in the environment and agri-food production system.
Project description:In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated. EMA-qPCR has been shown to be a promising rapid method for the detection of viable Campylobacter spp. from food samples. Application of membrane filtration and centrifugation, two methods frequently used for the isolation of bacteria from water, revealed a mean loss of up to 1.08 log10 cells/ml from spiked samples. Both methods used alone lead to a loss of dead bacteria and accumulation of viable bacteria in the sample as shown by fluorescence microscopy. After filtration of samples, no significant differences could be detected in subsequent qPCR experiments with and without EMA pretreatment compared to culture-based enumeration. High correlations (R(2)= 0.942 without EMA, R(2) = 0.893 with EMA) were obtained. After centrifugation of samples, qPCR results overestimated Campylobacter counts, whereas results from both EMA-qPCR and the reference method were comparable. As up to 81.59% of nonviable cells were detected in pond water, EMA-qPCR failed to detect correct quantities of viable cells. However, analyses of spiked tap water samples revealed a high correlation (R(2) = 0.863) between results from EMA-qPCR and the reference method. After membrane filtration, EMA-qPCR was successfully applied to Campylobacter field isolates, and results indicated an advantage over qPCR by analysing defined mixtures of viable and nonviable cells. In conclusion, EMA-qPCR is a suitable method to detect viable Campylobacter from water samples, but the isolation technique and the type/quality of the water sample impact the results.
Project description:The present study was conducted to determine whether recombinant human ?-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.
Project description:The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.