scholarly journals Colorimetric Point-of-Care Detection of Clostridium tyrobutyricum Spores in Milk Samples

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 293
Author(s):  
Paola Cecere ◽  
Francesca Gatto ◽  
Claudia Cortimiglia ◽  
Daniela Bassi ◽  
Franco Lucchini ◽  
...  
Keyword(s):  
Quality Control ◽  
Dna Extraction ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Raw Milk ◽  
Site Quality ◽  
Contamination Level ◽  
Clostridium Tyrobutyricum ◽  
Milk Samples ◽  
Point Of Care Detection

Clostridium tyrobutyricum represents the main spoiling agent responsible for late blowing defects (LBD) in hard and semi-hard cheeses. Its spores are resistant to manufacturing procedures and can germinate during the long ripening process, causing the burst of the cheese paste with a consequent undesirable taste. The lower quality of blown cheeses leads to considerable financial losses for the producers. The early identification of spore contaminations in raw milk samples thus assumes a pivotal role in industrial quality control. Herein, we developed a point of care (POC) testing method for the sensitive detection of C. tyrobutyricum in milk samples, combining fast DNA extraction (with no purification steps) with a robust colorimetric loop-mediated isothermal amplification (LAMP) technique. Our approach allows for the sensitive and specific detection of C. tyrobutyricum spores (limit of detection, LoD: ~2 spores/mL), with the advantage of a clear naked-eye visualization of the results and a potential semi-quantitative discrimination of the contamination level. In addition, we demonstrated the feasibility of this strategy using a portable battery-operated device that allowed both DNA extraction and amplification steps, proving its potential for on-site quality control applications without the requirement of sophisticated instrumentation and trained personnel.

scholarly journals Toxoplasma gondii and pre-treatment protocols for polymerase chain reaction analysis of milk samples: a field trial in sheep from Southern Italy

2017 ◽  
Vol 6 (1) ◽  
Author(s):  
Alice Vismarra ◽  
Elena Barilli ◽  
Maura Miceli ◽  
Carlo Mangia ◽  
Cristina Bacci ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Southern Italy ◽  
Raw Milk ◽  
Type I ◽  
Treatment Protocols ◽  
Milk Samples ◽  
Pre Treatment

Toxoplasmosis is a zoonotic disease caused by the protozoan <em>Toxoplasma gondii</em>. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on <em>T. gondii</em> tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable PCR positivity. This protocol was then used to analyze milk samples form sheep from three different farms in southern Italy, including Real Time PCR for DNA quantification and PCR-RFLP for genotyping. The pre-treatment protocol using EDTA and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, Real Time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of <em>T. gondii</em> transmission through consumption of raw milk and its unpasteurized derivatives.

scholarly journals Function of Preliminary Incubation of Raw Milk Samples for Quality Control

1989 ◽  
Vol 72 (6) ◽  
pp. 1443-1445 ◽  
Author(s):  
R. Burt Maxcy ◽  
Michael B. Liewen
Keyword(s):  
Quality Control ◽  
Raw Milk ◽  
Milk Samples

scholarly journals The Occurrence of Aflatoxin M1 in Milk, soft cheese and yoghurt in Baghdad Province by Using ELISA Test

2014 ◽  
Vol 38 (2) ◽  
pp. 9-16
Author(s):  
Najim Hadi Najim
Keyword(s):  
Dairy Products ◽  
Raw Milk ◽  
Elisa Test ◽  
Mean Value ◽  
Aflatoxin M1 ◽  
Contamination Level ◽  
Milk Samples ◽  
White Cheese ◽  
The Mean ◽  
Milk And Dairy Products

     Milk and dairy products are fundamental components in the human diet and may be the principle way for entrance of Aflatoxin M1 (AFM1) in to the human body. All milk and dairy products samples were tested for the occurrence of AFM1 by the competitive ELISA technique. Out of 32 bovine raw milk samples that were collected from eight villages around Baghdad province, 32 samples (100 %) were contaminated with AFM1 ranging from 0.15 to 86.96ng/kg with mean value of 42.37±26.07 ng/kg, of which 17 samples were contaminated with concentrations < 50 ng/kg and 15 samples exceeded the maximum acceptable level of AFM1 in milk (50 ng/kg) imposed by the European legislation. The raw milk samples belonged to animals fed with composite and stored fodder as in Althahab Alabiadh, Radhwaniya and Fadhaliya villages had higher significantly AFM1 concentrations over all the other five villages (Grazing feed). All 32 (100%) locally produced soft white cheese samples analyzed were contaminated with AFM1 ranging from 31.84 to 89.44 ng/kg with the mean value of 59.92±17.03 ng/kg. Out of 32 locally produced yoghurt samples analyzed, 32 samples (100%) were contaminated with AFM1 ranging from 0.16 to 42.74 ng/kg with the mean value of 16.92±11.55 ng/kg. Thirty samples (100%) of the examined 30 imported UHT milk samples that were collected from different commercial companies in the province of Baghdad presented significantly  high contamination level with AFM1 that were found to range from 0.18 to 85.66 ng/kg.

scholarly journals Presence of moulds and aflatoxin M1 in milk

2009 ◽  
pp. 63-68 ◽  
Author(s):  
Vesna Jankovic ◽  
Jelena Vukojevic ◽  
Brankica Lakicevic ◽  
Radmila Mitrovic ◽  
Dejan Vukovic
Keyword(s):  
Dairy Products ◽  
Raw Milk ◽  
Direct Result ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Contamination Level ◽  
Infant Food ◽  
Fungal Contamination ◽  
Milk Samples ◽  
Feed Samples

Aflatoxin M1 (AFM1) appears in milk or dairy products as a direct result of the cattle's ingestion of feed contaminated with aflatoxin B1 (AFB1). This study comprises mycological and mycotoxicological investigations of 23 milk samples (raw, infant food, pasteurized, whey and yoghurt). The mycological testing showed dominant presence of genus Geotrichum. G. candidum was found in 9 samples, with the highest contamination in the raw milk samples. The contamination level of AM1 is defined by using direct competitive enzyme- -linked immunosorbent assay (ELISA). AFM1 was found in 9 samples. AFM1 levels were lower than the recommended limits. However, as AFM1 is considered a probable human carcinogen (2B type), it is necessary to achieve a low level of AFM1 in milk. Therefore, cows' feed samples from various cowsheds are supposed to be evaluated routinely for aflatoxin, and kept away from fungal contamination as much as possible.

scholarly journals Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System

2016 ◽  
Vol 55 (1) ◽  
pp. 291-301 ◽  
Author(s):  
Padmapriya P. Banada ◽  
Srinidhi Deshpande ◽  
Soumitesh Chakravorty ◽  
Riccardo Russo ◽  
James Occi ◽  
...  
Keyword(s):  
Blood Culture ◽  
Francisella Tularensis ◽  
Whole Blood ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Bloodstream Infections ◽  
Content Type ◽  
Testing Procedures ◽  
Point Of Care Detection

ABSTRACTFrancisella tularensisis a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitiveF. tularensisassay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system.F. tularensisDNA in buffer or CFU ofF. tularensiswas spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected withF. tularensisSchu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/mlF. tularensisin both human and macaque blood. In infected macaques, the assay detectedF. tularensison days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detectF. tularensisin bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.

scholarly journals Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32

2022 ◽  
Vol 16 (1) ◽  
pp. e0010112
Author(s):  
Sirawit Jirawannaporn ◽  
Umaporn Limothai ◽  
Sasipha Tachaboon ◽  
Janejira Dinhuzen ◽  
Patcharakorn Kiatamornrak ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Clinical Performance ◽  
Point Of Care ◽  
Limited Resource ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Detection Assay ◽  
Short Palindromic Repeat ◽  
Flow Detection ◽  
Point Of Care Detection

Background One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. Methodology/Principal findings A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4–6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. Conclusions/Significance The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.

scholarly journals AFLATOXIN EXCRETION THROUGH MILK

2008 ◽  
Vol 1 (1) ◽  
pp. 66-70
Author(s):  
Milica Živkov Baloš ◽  
Željko Mihaljev ◽  
Mira Kovačević ◽  
Dejan Bugarski
Keyword(s):  
Thin Layer ◽  
Limit Of Detection ◽  
Dairy Farms ◽  
Raw Milk ◽  
Aflatoxin M1 ◽  
Elisa Method ◽  
Obligatory Control ◽  
Milk Samples ◽  
Two Samples ◽  
Layer Chromatography

In the period January to June 2006 the samples of feed were collected from feed factories in Southern Baåka and Srem district. The samples of raw milk and full mix were taken from 5 dairy farms. A total of 50 raw milk samples was examined. The samples were examined on the presence of aflatoxin B1 using the method of thin layer chromatography (TLC) and simultaneously, using ELISA tests. Milk samples were examined using immunoenzyme tests for the presence of aflatoxin M1. Aflatoxin content in all the examined feed and mix samples was below LOD (limit of detection) of TLC method, also this content was below MRL according to ELISA method. In total of 50 samples of raw milk, aflatoxin M1 was detected in two samples originating from different farms. Aflatoxin was detected in 7.5 ng/l, i.e. 10 ng/l respectively, what is considerably lower than MRL. Based on the obtained results it is considered that obligatory control of raw milk for the presence of aflatoxin is necessary.

A Novel Rapid Enzyme Immunoassay (Fluorophos BetaScreen) for Detection of β-Lactam Residues in Ex-Farm Raw Milk

1998 ◽  
Vol 61 (7) ◽  
pp. 808-811 ◽  
Author(s):  
ÅSE STERNESJÖ ◽  
GÖRAN JOHNSSON
Keyword(s):  
Enzyme Immunoassay ◽  
Limit Of Detection ◽  
Raw Milk ◽  
Penicillin G ◽  
Milk Samples ◽  
Quality Testing ◽  
Response Profile ◽  
Very High ◽  
Higher Sensitivity

A novel, qualitative enzyme immunoassay based on fluorescence detection for determination of (β-lactam antibiotics in raw, commingled milk (Fluorophos BetaScreen E. U. test) was evaluated. A dose-response profile for penicillin G was constructed by analysis of spiked milk samples. The limit of detection, defined as the concentration of penicillin G that resulted in 95% of the samples being evaluated as positive, was 1.8 μg/kg. The repeatability of the assay was very high both within and between the three participating milk quality testing laboratories. In total 5,061 randomly selected tanker milk samples were analyzed with the BetaScreen test and compared with the Delvotest SP. The agreement between the two tests was 99.7%. Probably due to a higher sensitivity to penicillin G, the BetaScreen test detected almost twice as many suspect positive tanker milk samples (0.45%) as the Delvotest SP (0.26%).

scholarly journals Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits—Application in Raw Milk Samples from Various Regions of Greece

2021 ◽  
Vol 8 (3) ◽  
pp. 46
Author(s):  
Martha Maggira ◽  
Maria Ioannidou ◽  
Ioannis Sakaridis ◽  
Georgios Samouris
Keyword(s):  
Limit Of Detection ◽  
Reference Method ◽  
Raw Milk ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fluorescence Detector ◽  
Elisa Kit ◽  
Milk Samples ◽  
Of Greece

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg−1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow’s, goat’s, and sheep’s milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk.

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