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1.
Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese 总被引:31,自引:0,他引:31
K Wernars C J Heuvelman T Chakraborty S H Notermans 《The Journal of applied bacteriology》1991,70(2):121-126
The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth 18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10(3) cfu/0.5g could be visualized whereas in others the presence of 10(8) cfu/0.5 g did not yield a detectable quantity of amplified product. 相似文献
2.
E. KACLÍKOVÁ K. ORAVCOVÁ T. KUCHTA D. PANGALLO 《Journal of Rapid Methods and Automation in Microbiology》2004,12(2):107-113
Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 100 cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 109 cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 106 cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1. 相似文献
3.
Kamtin Leung Michael B. Cassidy Steve B. Holmes Hung Lee Jack T. Trevors 《FEMS microbiology ecology》1995,16(1):71-82
Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g−1 of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 109 cfu g−1 of dry soil to below the detection threshold of both selective (14 cfu g−1 of dry soil) and nonselective (1 × 103 cfu g−1 of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 106 to 2.9 × 108 cfu g−1 of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 106 cfu g−1 of dry soil after the freeze-thaw treatment. 相似文献
4.
A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes 总被引:1,自引:0,他引:1
Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3'region of L. monocytogenes hly A gene spanning a conserved Hin dIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 104 cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10–100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days. 相似文献
5.
Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine 总被引:7,自引:2,他引:5
G. Zapparoli S. Torriani P. Pesente & F. Dellaglio 《Letters in applied microbiology》1998,27(5):243-246
Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 103 cfu ml−1 of oenococci were detected in fermenting grape must containing 107 yeast cells, whereas the detection limit in wine was 104 cfu ml−1 . The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control. 相似文献
6.
Abstract: A combined polymerase chain reaction amplification and reverse dot blot assay was designed for the detection of bacterial genes from soil and sediments. Total nucleic acids were directly extracted from soil using a lysozyme/sodium dodecyl sulfate/freeze-thaw method followed by rapid purification through gel electrophoresis. DNA was amplified using a highly stringent polymerase chain reaction with primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting amplification product was detected colorimetrically by reverse dot blot with an improved sensitivity ten-fold greater than traditional ethidium bromide staining after gel electrophersis. A lower limit of 103 , P. putida G7 cfu (g soil)−1 was detected. This method was successfully employed to detect indigenous naphthalene-degrading bacteria from subsurface sediment collected from different locations of a naphthalene-contaminated site. Similar approaches could be developed for other soil-borne genetic markers. 相似文献
7.
L. DOMINGUEZ J. F. F. GARAYZABAL J. A. VAZQUEZ J. L. BLANCO G. SUAREZ 《Letters in applied microbiology》1987,4(6):125-127
Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 105 Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 103 L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese. 相似文献
8.
The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes 总被引:2,自引:0,他引:2
The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin® pre-production to the milk. Each batch of cheese was inoculated with 102 –103 cfu g−1 of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l−1 could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss. 相似文献
9.
Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks 总被引:1,自引:0,他引:1
Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 106 cfu ml-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 106 cfu ml-1 ) could not survive the current HTST plate pasteurization protocol. 相似文献
10.
RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION 总被引:1,自引:0,他引:1
LIJU YANG CHUANMIN RUAN YANBIN LI 《Journal of Rapid Methods and Automation in Microbiology》2001,9(4):229-240
A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 103 105 cfu/mL, with R2 = 0.99. The detection limit of this method was 1.09 × 103 cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples. 相似文献
11.
Detection of bacterial antigens using immuno-PCR 总被引:4,自引:0,他引:4
E. Kakizaki T. Yoshida H. Kawakami M. Oseto T. Sakai M. Sakai 《Letters in applied microbiology》1996,23(2):101-103
E. KAKIZAKI, T. YOSHIDA, H. KAWAKAMI, M. OSETO, T. SAKAI AND M. SAKAI. 1996. A new and very sensitive antigen detection technique, immuno-polymerase chain reaction (immuno-PCR), was developed. This method is basically similar to the enzyme-linked immunosorbent assay which detects an antigen-antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida , in naturally infected yellowtail. Using immuno-PCR, 3.4 cfu ml−1 of bacteria could be detected. In comparison, ELISA detected only 3.4 × 104 cfu ml−1 . Immuno-PCR is a powerful method for detection of pathogens in host tissues. 相似文献
12.
E.F. DALEY J.M. FARBER D.W. WARBURTON 《Journal of Rapid Methods and Automation in Microbiology》1997,5(3):193-203
A direct plating method for the enumeration of low levels of foodborne Listeria monocytogenes was evaluated in a collaborative study involving 18 laboratories across Canada. Shrimp, coleslaw, ice cream and wieners were inoculated with low levels (5 × 102 and 103 /g) of L. monocytogenes and shipped to participants. Foods were diluted and then plated onto either lithium chloride phenylethyl and moxa-lactam agar (LPM), Oxford agar (OXA), modified Oxford agar (MOX) or Palcam agar (PAL). Recovery was good for all foods, except coleslaw. Of the four plating media tested, all were more or less equivalent in their ability to recover colonies for enumeration, except that more colonies were enumerated on LPM than on PAL agar. Recovery of L. monocytogenes ranged from <50 to 1250 cfu/g for wieners, <50 to 800 cfu/g for shrimp, <100 to 1440 cfu/g for ice cream and <50 to 700 cfu/g for coleslaw. Results indicate that the direct plating method can be used for the recovery of low levels of Listeria monocytogenes in Category 3 foods, as presently suggested for use in the Canadian Listeria compliance guide. 相似文献
13.
A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 102 –3·4 × 103 cfu ml−1 (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 102 –1·9 × 104 cfu per microplant sample (equivalent to 5 cfu – 3·8 × 102 cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material. 相似文献
14.
In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH2 PO4 , 2; NaHPO4 , 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×107 and 1×108 cells ml−1 with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×106 cells ml−1 when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×107 cfu ml−1 and 1×108 cfu ml−1 with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×105 cfu ml−1 in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope. 相似文献
15.
Susceptibility and resistance of anaerobic rumen fungi to antimicrobial feed additives 总被引:1,自引:0,他引:1
Listeria monocytogenes survived in meat, cheese and egg ravioli stored at 5°C for 14 d. Ravioli were considered edible for the first 9 d of storage. Initial L. monocytogenes populations of 3 × 105 cfu/g of ravioli were reduced to non-detectable levels after heat treatment simulating that which would be used by the consumer. 相似文献
16.
17.
Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive 总被引:2,自引:0,他引:2
The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–103 cfu g-1 than on leaves inoculated with about 105 cfu g-1 , but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested. 相似文献
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10
18.
Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk 总被引:2,自引:1,他引:1
A. Meyrand S. Boutrand-Loei S. Ray-Gueniot C. Mazuy C.E. Gaspard G. Jaubert G. Perrin C. Lapeyre & C. Vernozy-Rozand 《Journal of applied microbiology》1998,85(3):537-544
Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml−1 . Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 103 cfu ml−1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3·2 ng g−1 of cheese made with an initial population of 103 –106 cfu ml−1 . No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study. 相似文献
19.
A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk 总被引:1,自引:0,他引:1
R. Gutiérrez T. García I. González B. Sanz P.E. Hernández & R. Martín 《Journal of applied microbiology》1997,83(4):518-523
We have developed a quantitative PCR-ELISA for the rapid enumeration of bacteria inrefrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNAgene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment froma wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCRproducts generated using a digoxigenin-labelled primer were heat-denatured before beingquantified by an enzyme-linked immunosorbent assay (ELISA). A biotinylated probe immobilizedonto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments thatwere detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion ofsubstrate gave distinct absorbence differences when assaying milk samples containing bacteria inthe range 103 –107 cfu ml−1 . The detection threshold for thePCR-ELISA assay developed in this work is 103 cfu ml−1 . 相似文献
20.
Katrin Wendt-Potthoff Horst Backhaus Kornelia Smalla 《FEMS microbiology ecology》1994,15(3-4):279-290
Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×108 to 3.1×102 cfu g−1 on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×104 cfu g−1 (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×103 and 4×106 cfu g−1 fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment. 相似文献