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Results for “Immunomagnetic Separation”

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PubMed2001

Automated immunomagnetic separation and microarray detection of E. coli O157:H7 from poultry carcass rinse.

We describe the development and application of an electromagnetic flow cell and fluidics system for automated immunomagnetic separation (IMS) of Escherichia coli O157:H7 directly from poultry carcass rinse. We further describe the biochemical coupling of automated sample preparation with nucleic acid microarrays. Both the cell concentration system and microarray detection method did not require cell growth or enrichment from the poultry carcass rinse prior to IMS. Highly porous Ni foam was used to enhance the magnetic field gradient within the flow path, providing a mechanism for immobilizing immunomagnetic particles throughout the fluid rather than the tubing wall. A maximum of 32% recovery efficiency of non-pathogenic E. coli was achieved within the automated system with 6 s cell contact times using commercially available antibodies targeted against the O and K antigens. A 15-min protocol (from sample injection though elution) provided a cell recovery efficiency that was statistically similar to > I h batch captures. O157:H7 cells were reproducibly isolated directly from poultry carcass rinse with 39% recovery efficiency at 10(3) CFU ml(-1) inoculum. Direct plating of washed beads showed positive recovery of O157:H7 directly from poultry carcass rinse at an inoculum of 10 CFU ml(-1). Recovered beads were used for direct polymerase chain reaction (PCR) amplification and microarray detection, with a process-level detection limit (automated cell concentration though microarray detection) of < 10(3)CFU ml(-1) in poultry carcass rinse.

Animals↗

PubMed2002

Effects of pH and magnetic material on immunomagnetic separation of Cryptosporidium oocysts from concentrated water samples.

In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.

Animals↗

PubMed2003

Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes.

Four polyclonal anti-Listeria antibodies were evaluated for the detection of Listeria monocytogenes in direct and indirect assays using immunomagnetic separation with flow cytometry. The efficiency of immunocapturing using magnetic beads was also determined. None of the tested antibodies exhibited sufficient specificity or avidity to allow sufficient separation and detection of L. monocytogenes for a useful test that differentiated between negative (without cell) and positive (with cell) samples. Plating results confirmed that cells were captured with Dynabeads anti-Listeria and magnetic beads coated with goat anti-Listeria antibody with recovery ranging from 7 to 23%. Fluorescent-labeled polyclonal antibodies used in this study were not sufficiently specific to allow the detection of L. monocytogenes cells captured by the beads.

Animals↗

PubMed1995

The detection of Salmonella in skimmed milk powder enrichments using conventional methods and immunomagnetic separation.

Skimmed milk powders were spiked with one of three Salmonella serovars and incubated in buffered peptone water for 24 h. No false-negative results were obtained by immunomagnetic separation (IMS), compared to seven for selenite cysteine, one for Müller-Kauffmann tetrathionate and two for Rappaport-Vassiliadis enrichment broths. Salmonella virchow was detected and enumerated during the pre-enrichment incubation by IMS and indirect conductance techniques. The Salm. virchow cell number did not increase after 12 h incubation and remained at 3 x 10(6) cfu ml-1. IMS was able to capture Salm. virchow cells at cell numbers ca 50 ml-1 in the presence of a 1000 greater number of non-salmonella cells.

Animals↗

PubMed2003

Detection of Escherichia coli serogroups O26, O103, O111 and O145 from bovine faeces using immunomagnetic separation and PCR/DNA probe techniques.

AIMS: The aim of this study was to isolate Escherichia coli O26, O103, O111 and O145 from 745 samples of bovine faeces using (i) immunomagnetic separation (IMS) beads coated with antibodies to lipopolysaccharide, and slide agglutination (SA) tests and (ii) PCR and DNA probes for the detection of the Verocytotoxin (VT) genes. METHODS AND RESULTS: IMS-SA tests detected 132 isolates of presumptive E. coli O26, 112 (85%) were confirmed as serogroup O26 and 102 had the VT genes. One hundred and twenty-two strains of presumptive E. coli O103 were isolated by IMS-SA, 45 (37%) were confirmed as serogroup O103 but only one of these strains was identified as Verocytotoxin-producing E. coli (VTEC). Using the PCR/DNA probe method, 40 strains of VTEC O26 and three strains of VTEC O103 were isolated. IMS-SA identified 21 strains of presumptive E. coli O145, of which only four (19%) were confirmed as serogroup O145. VTEC of this serogroup was not detected by either IMS-SA or PCR/DNA probes. E. coli O111 was not isolated by either method. CONCLUSION: IMS beads were 2.5 times more sensitive than PCR/DNA probe methods for the detection of VTEC O26 in bovine faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: IMS-SA is a sensitive method for detecting specific E. coli serogroups. However, the specificity of this method would be enhanced by the introduction of selective media and the use of tube agglutination tests for confirmation of the preliminary SA results.

Agglutination Tests↗

PubMed1998

PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation.

The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.

AIDS-Related Opportunistic Infections↗

PubMed2004

Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces.

In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.

Animals↗

PubMed2006

Carbon nanotubes for immunomagnetic separation of Escherichia coli O157: H7.

Bovine serum albumin-functionalized multiple-walled carbon nanotubes with encapsulated ferromagnetic elements were conjugated with pathogen-specific antibody, and the conjugate was evaluated for immunomagnetic separation of Escherichia coli O157:H7 in pure and mixed (with Salmonella Typhimurium) cultures.

Coated Materials, Biocompatible↗

PubMed2004

Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation.

AIMS: To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. METHODS AND RESULTS: A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g-1 of faeces) to 2.4 x 104 MPN g-1. There was no significant difference (P = 0.06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g-1) than in pasture-fed (13 MPN g-1). CONCLUSIONS: Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies.

Animal Feed↗

PubMed1999

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry.

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.

Animals↗

PubMed1997

[Isolation of enterohemorrhagic Escherichia coli (O157:H7) by an immunomagnetic separation method].

Three sporadic cases of enterohemorrhagic Escherichia coli (EHEC) O157 infection which occurred in Kanagawa in 1996 were investigated. In an attempt to determine sources of the infection, a novel method of immunomagnetic separation (IMS) was employed to isolate the bacterium from feces, foods, and other associated items. In the first case, strains of EHEC O157:H7 producing Vero toxin (VT) 2 were isolated from both feces of the patient and suspected food (cattle liver) kept at a restaurant, and the strains were found to be genotypically identical through an analysis of pulsed-field gel electrophoresis (PFGE). Subsequent investigation in the meat processing store, from which the above cattle liver had been retailed to the restaurants revealed that the store was contaminated with EHEC O157:H7 producing both VT1 and VT2. In the second case, a strain isolated from the patient was EHEC O157:H7 producing both VT1 and VT2 while strains isolated from the patient's family (without apparent symptom) and the suspected facility were O137:NM producing VT2. PFGE analysis indicated that the latter two strains were genotypically identical, suggesting that the facility thus contaminated with EHEC O157 caused the infection in question. In the third case, EHEC O157:NM producing VT2 was isolated from 4 out of 7 family members including the patient, and these strains were found to be genotypically identical by subsequent PFGE analysis. Source of the infection was, however, not determined due to lack of suspected food items. In this context, four slaughterhouses in Kanagawa Prefecture were investigated for presence of EHEC O157. As a result, strains of EHEC O157:H7 producing VT1 and VT2 were isolated from the contents of cattle's distal colon and surface of the skinned carcasses. Additional attempt was also made to determine a possibility of river water being contaminated with EHEC O157. The bacterium was, however, not isolated from water samples collected from 4 major rivers in the prefecture (at 10 collecting sites). Experiments were undertaken in order to evaluate the use of IMS in isolation of EHEC O157 from food items, with different pre-enrichment media and conditions. The results indicated that pre-enrichment by trypticase soy broth at 36 degrees C for 6 h followed by inoculating onto sorbitol MacConkey agar plate containing cefixime and tellurite was most appropriate to isolate EHEC O157 strains.

Adolescent↗

PubMed2004

Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7.

A sensitive, specific, and rapid method for the detection of E. coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-E. coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E. coli antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer. The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E. coli O157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method. The total detection time was less than 2 h. Neither E. coli K12 nor Salmonella typhimurium interfered with the detection of E. coli O157:H7.

Escherichia coli O157↗

PubMed2001

Screening bovine carcass sponge samples for Escherichia coli O157 using a short enrichment coupled with immunomagnetic separation and a polymerase chain reaction-based (BAX) detection stept.

A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E. coli O157 polymerase chain reaction assay. This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples. Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at -20 degrees C, then subjected to BAX analysis. The results showed a high degree of agreement between the plating method and the BAX system. Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity). Of the 76 samples that appeared negative for the presence of E. coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity). Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis. Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E. coli O157:H7 in carcass sponge samples. Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.

Animals↗

PubMed1996

A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from cases of bloody diarrhoea, non-bloody diarrhoea and asymptomatic contacts.

Enrichment culture in modified buffered peptone water followed by immunomagnetic separation (IMS) with magnetic beads coated with an antibody against Escherichia coli O157 was compared with direct culture on cefixime rhamnose sorbitol MacConkey agar (CR-SMAC) and cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for the isolation of E. coli O157 from human faeces. In total, 690 samples were examined; E. coli O157 was isolated from 25 samples by IMS but from only 15 and 12 by direct culture on CT-SMAC and CR-SMAC, respectively. The difference in sensitivity of detection was at its most marked on screening repeat faecal samples from known cases and samples from asymptomatic contacts, when of 12 strains of E. coli O157 isolated by IMS, only five were isolated by direct culture. IMS is a sensitive and simple technique for the isolation of E. coli O157 from human faecal samples and should prove useful in elucidating further the epidemiology of this micro-organism.

Acute Disease↗

PubMed2003

Detection of pathogenic Listeria spp. in zoo animal faeces: use of immunomagnetic separation and a chromogenic isolation medium.

Faecal samples, collected from 200 healthy animals in Antwerp Zoo, were examined for the presence of pathogenic Listeria spp. A two-stage standard isolation (ISO) method was combined with immunomagnetic separation (IMS). ALOA agar, a chromogenic isolation medium, differentiating Listeria spp. on the basis of beta-glucosidase and phosphatidylinositol-specific phospholipase C (PIPLC) activity, was compared with PALCAM agar. Confirmation of the isolates was based on conventional biochemical tests and a disc test, which detects a specific aminopeptidase produced by all Listeria spp. except Listeria monocytogenes. Listeria spp. were isolated from 42 (21.0%), L. monocytogenes from 14 (7.0%), and Listeria ivanovii from two (1.0%) faecal samples. The application of IMS after primary enrichment detected pathogenic Listeria spp. in 12 (6.0%) samples. The ISO method, combining primary and secondary enrichment, detected pathogenic Listeria spp. in 15 (7.5%) samples. The sensitivity of IMS compared to the ISO method was 73.3% and the specificity was 99.5%. ALOA agar was superior to PALCAM agar for isolation of Listeria spp. The disc test identified all L. monocytogenes isolates. IMS after primary enrichment was a suitable screening method, but secondary enrichment increased the number of positive samples.

Animals↗

PubMed1996

Immunomagnetic separation and solid-phase detection of Bordetella pertussis.

In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.

Base Sequence↗

PubMed1997

Use of commercial enzyme immunoassays and immunomagnetic separation systems for detecting Escherichia coli O157 in bovine fecal samples.

A commercial enzyme immunoassay (EIA) (E. coli O157 Visual Immunoassay; Tecra Diagnostics) performed on enrichment cultures in modified Escherichia coli broth (mECn) was compared with immunomagnetic separation (IMS) (Dynabeads anti-E. coli O157; Dynal) performed on enrichment cultures in modified buffered peptone water (BPW-VCC) for the detection of E. coli O157 in bovine fecal samples. Tests on fecal suspensions inoculated with each of 12 different strains of E. coli O157 showed that both the EIA and IMS methods were 10- to 100-fold more sensitive than direct culture or enrichment subculture methods for detection of the organism. EIA and IMS were then compared for detection of E. coli O157 in bovine rectal swabs. For confirmation of positive EIA tests, a commercial system (Immunocapture System [ICS]; Tecra Diagnostics) was compared with IMS; both were performed on mECn enrichment cultures. Of 200 rectal swabs examined, 17 gave positive results in the EIA which were confirmed by both confirmation systems, 2 gave positive results in the EIA which were confirmed by IMS but not by ICS, and 1 gave a positive result in the EIA which was confirmed by ICS but not by IMS. Of these 20, 15 were also positive by the BPW-VCC-IMS culture system; a further 3 samples were positive by this culture system but gave a negative result in the EIA. Eight samples were negative by the BPW-VCC-IMS culture system but gave a positive result in the EIA which could not be confirmed by either confirmation system. Further examination of the eight unconfirmed EIA-positive samples yielded sorbitol-fermenting E. coli O157 from three samples. Of the remaining five cultures, four were positive in an EIA for verocytotoxins (VT) and two were positive in a cell culture assay for VT1. The remaining 170 samples were negative by both EIA and BPW-VCC-IMS. The Tecra EIA and IMS are both technically simple and sensitive methods for detecting E. coli O157 in bovine fecal samples. There was no statistically significant difference between the numbers of positives detected by the different assays (P = 0.29).

Animals↗

PubMed2003

New method using sedimentation and immunomagnetic separation for isolation and enumeration of Cryptosporidium parvum oocysts and Giardia lamblia cysts.

A new method for the isolation of Cryptosporidium parvum oocysts and Giardia lamblia cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (P < 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (P > 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.

Anaerobiosis↗