Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Association between respiratory and postural adaptations and self-perception of school-aged children with mouth breathing in relation to their quality of life

Objective: To investigate the respiratory and postural adaptations associated with mouth and nasal breathing and to evaluate the associations of such adaptations in mouth breathers' self-perceived quality of life. Method: Cross-sectional study with mouth breathers (initial n=116 and final n=48) and nasal breathers (initial n=131 and final n=24) from elementary school, aged between 7 and 14 years. Chest expansion, using cirtometry, the breathing pattern and the use of accessory muscles, by means of clinical evaluations and photogrammetry, and flexibility tests were evaluated in both groups. Subsequently, the mouth breathers were asked to complete the quality of life questionnaire. Statistical tests: Chi-square, odds ratio, Mann-Whitney, and binomial tests were first applied followed by logistic regressions. Results: Thoracic breathing (p=0.04), using of accessory muscles (p=0.03) and reductions in flexibility (p=0.001) increased the chances of an individual being a mouth breather when compared to nasal breathers. Subsequently, using of accessory muscles decreased the chances of snoring among mouth breathers (p=0.03); the presence of shoulder asymmetry reduced the chances of experiencing quiet sleep (p=0.05) and increased the chances of coughing or being tired when playing or running (p=0.008). Finally, forward head position reduced the chances of waking up at night (p=0.04) and experiencing shortness of breath (p=0.05). Conclusions: Respiratory and postural adaptations increased the chances of individuals persisting with mouth breathing. Additionally, these adaptations could be associated with mouth breathers' self-perceived quality of life. .

Proteomic profiling of nephrotic syndrome in serum using magnetic bead based sample fractionation & MALDI-TOF MS.

Background & objectives: At present, the diagnosis of nephrotic syndrome (NS) requires a renal biopsy which is an invasive procedure. We undertook this pilot study to develop an alternative method and potential new biomarkers for diagnosis, and validated a set of well-integrated tools called ClinProt to investigate serum petidome in NS patients. Methods: The fasting blood samples from 49 patients diagnosed with NS by renal biopsy, including 17 mesangial proliferative glomerulonephritis (MsPGN), 12 minimal change nephrotic syndrome (MCNS), 10 focal segmental glomerulosclerosis (FSGS) and 10 membranous nephropathy (MN), were collected and screened to describe their variability of the serum peptidome. The results in NS group were compared with those in 10 control healthy individuals. Specimens were purified with magnetic beads-based weak cation exchange chromatography and analyzed in a MALDI-TOF MS. Results: The results showed 43, 61, 45 and 19 differential peptide peaks in MsPGN, MCNS, MN and FSGS groups, respectively. A Genetic Algorithm was used to set up the classification models. Cross validation of healthy controls from MsPGN, MCNS, MN and FSGS was 96.18, 100, 98.53 and 94.12 per cent, respectively. The recognition capabilities were 100 per cent. Interpretation & conclusions: Our results showed that proteomic analysis of serum with MALDI-TOF MS is a fast and reproducible approach, which may give an early idea of the pathology of nephrotic syndrome.

Detección inmunomagnética de células tumorales circulantes en cáncer de mama metastásico: nuevas tecnologías / Inmunomagnetic detection of circulating tumor cells in metastatic breast cancer: new technologies

Las metástasis hematógenas son la mayor causa de mortalidad en el cáncer de mama. Está documentado que una vez las células tumorales se diseminan el resultado es, generalmente, letal. Las células tumorales circulantes han sido consideradas por largo tiempo un reflejo de la agresividad de los tumores, y entre ellos uno de los más agresivos es el cáncer de mama metastásico. Los primeros resultados clínicos han permitido determinar una fuerte relación entre la detección y el número de las células tumorales circulantes, como un valor pronóstico y como marcador de la actividad antitumoral del tratamiento. El análisis inmunomagnético utilizando una nueva metodología permite determinar que un recuento de 5 células tumorales circulantes o más en 7,5 ml de sangre, en cualquier fase de la enfermedad, se asocia a un mal pronóstico, y es predictivo de una supervivencia global más corta.

Hematogenous metastasis is the major cause of mortality in breast cancer. Evidence indicates that tumor cells escape from the primary tumor mass into the blood stream and that these disseminated cells are the source of increased lethality. Circulating or metastatic tumour cells have been considered as useful indicators of the aggressiveness of breast cancer tumours. The first clinical results obtained with such assays strongly suggest that in metastatic breast cancer, circulating tumour cells detection and enumeration can be used to estimate prognosis and may serve as an early marker to assess anti-tumour activity of a treatment. Immunomagnetic analysis using a new methodology, determine that a circulating tumour cells count of 5 or more per 7,5 ml of blood, at any time during the course of the disease is associated with a poor prognosis and is predictive of shorter progression and overall survival.

Evaluation of an immunomagnetic capture method followed by PCR to detect Mycobacterium bovis in tissue samples from cattle / Evaluación de un método de captura inmunomagnética seguida de PCR para la detección de Mycobacterium bovis en tejidos de ganado

Tuberculosis is one of the most important infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), an important animal pathogen with public health implications as it is a zoonosis. Currently, the diagnosis of BTB is based on the caudal fold test of the Tuberculin Skin Test (TST). Post-mortem bacterial culture is carried out to confirm the diagnosis, and then specific biochemical tests are performed for the characterization of the etiologic agent. Culture takes at least 4 to 8 weeks to develop. The diagnosis by molecular tests such as PCR can provide fast and reliable results, significantly decreasing the time of confirmation (from two months to two days), thus allowing the possibility of taking control actions to prevent the spread of the disease in herds. In this work the use of an immunomagnetic separation capture followed by PCR (IMS-PCR) based on the IS6110 element showed a detection threshold corresponding to 10 CFU in M. bovis-spiked PBS. In the case of infected bovine fresh tissues, after five replicates, the minimum value of detection was 1000 CFU in 100% of the trials (5/5). This paper attempts to provide a sensitive, rapid and specific technique for the diagnosis of bovine tuberculosis, and opens up the possibility of a direct application in the control and eradication of this cattle disease.

La tuberculosis es una de las enfermedades infecciosas más importantes. Mycobacterium bovis es el agente causal de la tuberculosis bovina (TBB), un patógeno animal y zoonótico. En la actualidad, el diagnóstico de TBB se basa en la prueba intradérmica de la tuberculina. El cultivo bacteriano post mortem se lleva a cabo para confirmar el diagnóstico y a continuación se realizan pruebas bioquímicas específicas para la caracterización del agente etiológico. El cultivo bacteriano toma por lo menos 4 a 8 semanas para su desarrollo. El diagnóstico mediante pruebas moleculares como PCR puede proporcionar resultados rápidos y robustos, con un considerable acortamiento hasta la confirmación del diagnóstico (de 2 meses a 2 días). En este trabajo, el uso de captura inmunomagnética seguida de PCR (IMS-PCR) dirigida al elemento IS6110 mostró un umbral de detección correspondiente a 10 UFC en M. bovis diluido en PBS. En el caso de tejidos bovinos inoculados experimentalmente después de 5 réplicas, el valor mínimo de detección fue de 1000 UFC en el 100% de los ensayos. Este artículo aspira a proporcionar una técnica sensible, rápida y específica para el diagnóstico de la tuberculosis bovina, con el fin de abrir la posibilidad de una aplicación directa en el control y la erradicación de esta enfermedad en el ganado.

Viabilidad de un aislado de Escherichia coli O157: H7 en queso tipo guayanés usando separación inmunomagnética como herramienta de recuperación / The viability of an Escherichia coli O157: H7 strain in guayanés Ccheese using immunomagnetic separation as a tool for recovery

Se determinó la viabilidad de Escherichia coli O157:H7 en queso guayanés de manufactura artesanal, evaluando distintos esquemas de aislamiento basados en separación inmunomagnética (SIM). Unidades de queso (25 g) fueron inoculadas con 25 y 250 cel/g del patógeno y almacenadas a 4°C. Las piezas se analizaron los días 0, 2, 6, 8 y 10 post-inoculación a través de distintos esquemas de separación inmunomagnética (SIM) que incluían dos caldos de enriquecimiento: agua de peptona buferada sin inhibidores (APB-SI) y agua de peptona buferada con vancomicina, cefixime y telurito (APB-VCT) y dos agares de aislamiento del inmunoseparado: agar MacConkey sorbitol (MCS) y agar MacConkey sorbitol con telurito y cefixime (MCS-TC). Los resultados demostraron la viabilidad del patógeno hasta por 10 días post-inoculación y en el transcurso de este tiempo, para algunos de los esquemas aplicados sobre la base de SIM, se logró un incremento en los porcentajes de recuperación, lo que indica que el número de células inoculadas se elevó con el tiempo. En cuanto a la utilidad de la SIM para la recuperación del patógeno, se observó variaciones en los porcentajes de aislamiento en función del caldo de enriquecimiento y el nivel de células inoculadas. Los mayores porcentajes de recuperación se obtuvieron en las piezas inoculadas con 250 cel/g, con rangos del 35 al 85 por ciento (día 0 y 10 respectivamente) en el mejor de los esquemas SIM (APB-SI/SIM/MCS), mientras que para niveles de 25 cel/g, en el mejor de los casos (APB-SI/SIM/MCS), durante los primeros 6 días no superó el 15 por ciento. El caldo de enriquecimiento de mejor desempeño fue APB-SI (p <0,05) y no se observó diferencias en los porcentajes de recuperación (p>0,05) en función de los agares utilizados (MCS y MCS-TC) para la siembra del inmunoseparado

The viability of an Escherichia coli O157:H7 strain in cottage-industry Guayanes cheese was determined by evaluating several isolation protocols based on immunomagnetic separation (IMS). Cheese units (25 g) were inoculated with 25 and 250 cel/g of this pathogen and stored at 4°C. The pieces were analized at 0, 2, 6, 8 and 10 days post-inoculation through several IMS protocols including two enrichment broths: buffered peptone water without inhibitors (BPW-WI) and buffered peptone water with vancomicyn, cefixime and telurite (BPW-VCT) and two immunoseparation isolation agars: MacConkey-sorbitol agar (MSA) and MacConkey-sorbitol agar with cefixime and telurite (MSA-CT). Results demonstrated the viability of the pathogen for up to 10 days post-inoculation, and during this time, for some of the schemes applied on the IMS base, an increase in recovery percentages was achieved, indicating that the number of inoculated cells increased with time. In terms of the utility of IMS for recovering the pathogen, variations in the isolation percentages were observed in terms of the enrichment broth and the level of inoculated cells. The biggest recovery percentages were obtained in pieces inoculated with 250 cel/g, with ranges between 35 and 85 percent (days 0 and 10 respectively) in the best IMS scheme (BPW-WI/IMS/MSA), while, at levels of 25 cel/g, in the best case (BPW-WI/IMS/MSA), 15 percent was not surpassed during the first six days. The best performing enrichment broth was BPW-WI (p<0.05) and differences in the recovery percentages (p>0.05) were not observed in relation to the agars (MSA and MSA-CT) used for sowing the immunoseparator

Cryptosporidium Oocyst Detection in Water Samples: Floatation Technique Enhanced with Immunofluorescence Is as Effective as Immunomagnetic Separation Method

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10(1), 10(2), and 10(3) per 10 microliter were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10(2) per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.

Examination of diarrheal stools in Hat Yai City, South Thailand, for Escherichia coli O157 and other diarrheagenic Escherichia coli using immunomagnetic separation and PCR method.

A total of 493 stool samples from diarrheal patients in Songklanagarind Hospital, in southern Thailand, were examined for Escherichia coli O157 by the culture method combined with an immunomagnetic separation (IMS) technique. E. coli O157 was not found, although the IMS-based method could detect 10(2)-10(3) CFU of artificially inoculated O157/g of stool samples. Polymerase chain reaction was also used for the detection and identification of diarrheagenic E coli from 530 stool samples. The target genes were eae for enteropathogenic E. coli (EPEC), stx for enterohemorrhagic E. coli (EHEC), elt and est for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC), and aggR for enteroaggregative E. coli (EAggEC). Fifty-eight diarrheagenic E. coli strains were detected in 55 stool samples (10%) from 32 children and 23 adults. These included 31 EAggEC strains (5.8%), 13 ETEC strains (2.5%), 13 EPEC strains (2.5%), and one EIEC strain (0.2%). EHEC was not detected. The diarrheagenic E. coli strains were found mainly in children under 2 years of age (24 of 32 children). EAggEC strains and ETEC strains were susceptible to several antibiotics whereas the EPEC strains exhibited resistance to these antibiotics.

A Comparative Study of Magnetic-Activated Cell Sorting, Cytotoxicity and Preplating for the Purification of Human Myoblasts

Although cultured myoblast transplantation has been extensively studied as a gene complementation approach to muscular dystrophy treatment, clinical success has still been limited. The inability to adequately isolate and purify myoblasts presents a major limitation to the production of sufficient myoblasts for engrafting purposes. This study attempted to purify myoblasts from primary culture by magnetic-activated cell sorting (MACS), complement-mediated cytotoxicity, and a preplating technique. As a result of positive myoblasts selection by MACS, the average percentage of myoblasts in mixed culture was increased from 30.0% to 41.7%. We observed both myoblast lysis and fibroblast lysis after complement-mediated cytotoxicity. Enrichment of myoblasts in mixed culture was found to increase to 83.1% by using the preplating technique. In addition, higher purification (92.8%) was achieved by following the preplating technique with MACS. Thus, preplating in combination with magnetic-activated cell sorting allows for a rapid and effective isolation of myoblasts from human muscle tissue.

Utilization of immunomagnetic separation for detection of Salmonella in raw broiler parts

This study was conducted aiming to compare the conventional microbiological method to detect Salmonella in broiler parts with the Immunomagnetic Separation method(IMS) followed by plate isolation and also the IMS associated with Rappaport-Vassiliadis broth (RV). The IMS was performed following a pre-enrichment step in buffered peptone water. Sixty-one samples (raw broiler parts) were tested and the results showed that the use of the IMS method allowed the isolation of Salmonella in 9 of the tested samples, while the association IMS/RV detected the agent in 30 samples. The conventional microbiological method was able to isolate the agent in 25 opportunities. These results allowed to conclude that the IMS/RV association presented an increased sensitivity and permitted a better isolation of Salmonella. The conclusion was that other means of isolation, in particular those which do not interfere with the growth of bead bounded Salmonella, should be searched.