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 Isolation and molecular characterization of Mycobacterium tuberculosis complex isolated from raw milk in some dairy farms in Egypt

2016 ◽  
Vol 5 (2) ◽  
pp. 105
Author(s):  
Heba Hussien ◽  
Eman Mahrous
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Raw Milk ◽  
Accurate Method ◽  
Tuberculosis Complex ◽  
Milk Samples ◽  
Source Of Infection

<p>This study was conducted to detect <em>Mycobacterium tuberculosis</em> complex in milk in three Egyptian Governorates; El-Sharkia, El-Menoufia and El-Behera Governorates. 300 milk samples were collected from tuberculin positive cases, 18 (6.0%) were shedding <em>Mycobacterium tuberculosis</em> complex in their milk which detected by real time PCR. On another hand, 170 milk samples were collected from tuberculin negative cases, 5 (2.9%) were shedding <em>Mycobacterium tuberculosis</em> complex in their milk which detected by real time PCR. All milk samples were examined by three techniques including Microscopic examination, culture and real time PCR. Real time PCR is more rapid and accurate method than microscopic and culture method. The isolated colonies from culture were examined by Multiplex PCR to demonstrate the source of infection either human or animal source.</p>

scholarly journals Identification of Coxiella burnetii in Raw Milk of Livestock Animal in Iran

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Ashraf Mohabati Mobarez ◽  
Ehsan Mostafavi ◽  
Mohammad Khalili ◽  
Saber Esmaeili
Keyword(s):  
Real Time ◽  
Coxiella Burnetii ◽  
Real Time Pcr ◽  
Q Fever ◽  
Goat Milk ◽  
Raw Milk ◽  
Molecular Evidence ◽  
Milk Samples ◽  
Sheep Milk ◽  
Dairy Animals

Coxiella burnetii is the causative agent of Q fever in humans and animals. This study aimed to determine the frequency of C. burnetii in milk samples of dairy animals (goats, sheep, and cattle) in some selected regions in Iran, where there is no information about prevalence of C. burnetii. In this study, 162 individual milk samples were collected from 43 farms in three provinces (Tehran, Hamadan, and Mazandaran). Real-time PCR was used for the detection of IS1111a element of C. burnetii. In total, 23 of 162 samples (14.2%, 95% confidence interval (CI): 9.65–20.2%) were positive for C. burnetii by real-time PCR. C. burnetii was detected in 10.17% (95% CI: 4.74–20.46) of goat milk samples. In sheep milk samples, 18.6% (95% CI: 9.74–32.62) were positive, and C. burnetii was detected in 15% (95% CI: 8.1–26.11) of cattle milk samples. Molecular evidence of the presence of C. burnetii was seen in milk samples of dairy animals in all the studied regions. These findings demonstrated that C. burnetii infection, especially in raw milk samples, deserves more attention from the health care system and veterinary organization in Iran.

Detection of staphylococcal enterotoxin genes in raw milk samples by real-time PCR

2019 ◽  
Author(s):  
Akýn Yiðin ◽  
Mehmet Demirci ◽  
Serap Kýlýç Altun ◽  
Hikmet Dinç
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Public Health Problem ◽  
Raw Milk ◽  
Staphylococcal Enterotoxin ◽  
Donkey Milk ◽  
Milk Samples ◽  
Conventional Culture ◽  
Food Borne ◽  
Enterotoxin Genes

Presence of significant level of enterotoxigenic S. aureus in raw milk of sheep, goat and donkey may cause serious food borne disease. Many people worldwide, use raw milk in their daily life but data on presence of virulence genes in cow, sheep, goat and especially donkey milk seems to be very limited. For this reason, aim of this study was to determine the presence both S. aureus and nine staphylococcal enterotoxin genes in cow, sheep, goat and donkey milks. A total of 231 raw milk samples were collected from 48 cow, 65 goat, 65 sheep and 53 donkey were collected. To detect presence of S. aureus both conventional culture and real-time PCR were used and to detect nine staphylococcal enterotoxin genes directly in milk, real-time PCR was performed. Conventional culture and real-time PCR results were found to be similar for presence of S. aureus and it was detected in 52 (22.51%) out of 231 raw milk samples. Staphylococcal enterotoxin genes were detected in 27 out of 52 S.aureus positive samples and a total of 62 enterotoxin genes were detected in these samples. However enterotoxin genes could not be detected in two S. aureus positive donkey milk. Hence, real-time PCR proved to be reliable and faster than conventional methods to detect presence of enterotoxigenic S. aureus in milk. Raw milk samples from different animals many contain enterotoxigenic S. aureus. Therefore, one should be careful during raw milk consumption as enterotoxigenic S. aureus in milk may cause dangerous public health problem which need routine screening for this pathogen in milk

The first detection ofToxoplasma gondiiDNA in environmental air samples using gelatine filters, real-time PCR and loop-mediated isothermal (LAMP) assays: qualitative and quantitative analysis

Parasitology ◽  
2017 ◽  
Vol 144 (13) ◽  
pp. 1791-1801 ◽  
Author(s):  
ANNA LASS ◽  
BEATA SZOSTAKOWSKA ◽  
KRZYSZTOF KORZENIEWSKI ◽  
PANAGIOTIS KARANIS
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Microscopic Analysis ◽  
Detection Methods ◽  
Type I ◽  
Air Samples ◽  
Two Samples ◽  
Environmental Air ◽  
Tract Infections

SUMMARYToxoplasma gondiiinfections are acquired through the ingestion of oocysts present in the environment. However, there is no data about their occurrence in the air or about airborne transmission of these infections. In the present paper, we report on the identification ofT. gondiiusing rapid molecular detection methods, supported by microscopic analysis, in environmental air samples. A total of 71 samples were collected, using gelatine filters, from kitchen gardens, recreational areas and sandpits located in northern and north-eastern Poland. Material recovered from the filters was analysed using real-time PCR and loop-mediated isothermal assays targeting theT. gondiiB1 gene.Toxoplasma gondiiDNA was found in two samples, as confirmed by both molecular assays. Genotyping at the SAG2 locus showedToxoplasmaSAG2 type I. Moreover, the presence ofT. gondiioocysts was confirmed in one of the positive samples with the use of microscopy. The results showed thatT. gondiimay be present in environmental air samples and that respiratory tract infections may play a role in the high prevalence of toxoplasmosis in humans and animals. To the best of our knowledge, this is the first epidemiological evidence that oro-fecal and foodborne toxoplasmosis may be traceable to an airborne respiratory origin and that this may represent a new, previously unknown transmission route for this disease.

Application of an F57 Sequence-Based Real-Time PCR Assay for Mycobacterium paratuberculosis Detection in Bulk Tank Raw Milk and Slaughtered Healthy Dairy Cows

2006 ◽  
Vol 69 (7) ◽  
pp. 1662-1667 ◽  
Author(s):  
C. BOSSHARD ◽  
R. STEPHAN ◽  
T. TASARA
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Raw Milk ◽  
Mesenteric Lymph Nodes ◽  
Pcr Assay ◽  
Milk Samples ◽  
Light Cycler ◽  
Bulk Tank ◽  
Mycobacterium Avium Subsp Paratuberculosis

A light cycler–based real-time PCR assay that targets the F57 sequence was used to collect data on the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in 100 bulk tank raw milk samples and in a population of 101 slaughtered dairy cattle. The assay's reproducible detection limit in total genomic DNA templates isolated from 10-ml samples of MAP-spiked raw milk was 100 cells per ml. Similarly, the evaluation of MAP-spiked bovine feces also demonstrated that the assay had a reproducible detection limit of 100 cells if they were contained within 200 mg of fecal sample material. Among the 100 bulk tank milk samples that were tested, we found 3 samples (3%) to be positive for MAP. In the slaughterhouse part of the study, 8.9% (9 of 101) of the cows were positive for MAP DNA in fecal samples, 4.9% (5 of 101) in mesenteric lymph nodes, 0.9% (1 of 101) in ileum tissue, and 3.6% (3 of 84) in milk. Meanwhile, for 2.9% (3 of 101) of the culled cows, MAP DNA was detected in samples of diaphragmatic muscles.

scholarly journals First molecular detection of Toxoplasma gondii in vegetable samples in China using qualitative, quantitative real-time PCR and multilocus genotyping

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anna Lass ◽  
Liqing Ma ◽  
Ioannis Kontogeorgos ◽  
Xueyong Zhang ◽  
Xiuping Li ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Tibet Plateau ◽  
Type I ◽  
Quantitative Real Time Pcr ◽  
Vegetable Samples ◽  
Multilocus Genotyping ◽  
Fresh Vegetables ◽  
Open Markets

AbstractToxoplasma gondii infection is becoming increasing problem in China but there is no data concerning contamination of vegetables intended for consumption with this parasite. The aim of the present study was to investigate fresh vegetables originated from open markets located in the Xining City, the Qinghai-Tibet Plateau (QTP), P.R. China for their contamination with T. gondii. A total of 279 fresh vegetable samples were collected and analysed using real-time PCR assay targeting B1 gene and multilocus genotyping. T. gondii DNA was found in 10 (3.6%) samples tested; eight of them represented T. gondii type I and remaining two T. gondii type II. The approximate level of contamination of positive vegetables samples, estimated based on quantitative real-time PCR (qPCR), ranged between less than one and 27000 T. gondii oocysts per sample, with majority not exceeding several oocysts per sample. The results of the study confirmed that T. gondii is present in vegetables offered in open markets in the Qinghai province, P.R. China; eating them unwashed and raw may therefore pose a threat to consumers. This is the first investigation describing T. gondii detection in fresh vegetables intended for consumption collected from the territory of P.R. China using sensitive molecular tools.

scholarly journals Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

2012 ◽  
Vol 78 (16) ◽  
pp. 5855-5863 ◽  
Author(s):  
Matthew L. Ranieri ◽  
Reid A. Ivy ◽  
W. Robert Mitchell ◽  
Emma Call ◽  
Stephanie N. Masiello ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Raw Milk ◽  
Processing Plant ◽  
Content Type ◽  
Milk Samples ◽  
Heat Treated ◽  
Spoilage Bacteria ◽  
Taqman Pcr ◽  
Fluid Milk

ABSTRACTPsychrotolerant sporeformers, specificallyPaenibacillusspp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. WhilePaenibacillusspp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number ofPaenibacillusspp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detectPaenibacillusspp. in fluid milk and to discriminate betweenPaenibacillusand other closely related spore-forming bacteria. Specificity was confirmed using 16Paenibacillusand 17Bacillusisolates. All 16Paenibacillusisolates were detected with a mean cycle threshold (CT) of 19.14 ± 0.54. While 14/17Bacillusisolates showed no signal (CT> 40), 3Bacillusisolates showed very weak positive signals (CT= 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 101CFU/ml using total genomic DNA extracted from raw milk samples inoculated withPaenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection ofPaenibacillus. Heat-treated milk samples wherePaenibacillus(≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection ofPaenibacillusthat has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.

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