Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. 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 T. gondii 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 T. gondii transmission through consumption of raw milk and its unpasteurized derivatives.

Download Full-text

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

International Journal of Basic and Applied Sciences ◽

10.14419/ijbas.v5i2.5299 ◽

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

This study was conducted to detect Mycobacterium tuberculosis 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 Mycobacterium tuberculosis 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 Mycobacterium tuberculosis 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.

Download Full-text

Development and application of a new multiplex real-time PCR assay for simultaneous identification of Brucella melitensis , Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

International Journal of Dairy Technology ◽

10.1111/1471-0307.12500 ◽

2018 ◽

Vol 71 (3) ◽

pp. 629-636 ◽

Cited By ~ 3

Author(s):

Esen Tutar ◽

Kübra Sueda Akıncı ◽

İsmaİl Akyol

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Brucella Melitensis ◽

Raw Milk ◽

Cronobacter Sakazakii ◽

Pcr Assay ◽

Simultaneous Identification

Download Full-text

Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR

Acta Veterinaria Hungarica ◽

10.1556/avet.2014.013 ◽

2014 ◽

Vol 62 (3) ◽

pp. 304-316 ◽

Cited By ~ 1

Author(s):

Orsolya Erdősi ◽

Katalin Szakmár ◽

Olivér Reichart ◽

Zsuzsanna Szili ◽

Noémi László ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Redox Potential ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Raw Milk ◽

Food Samples ◽

Potential Measurement ◽

Effective Manner ◽

Soft Cheese

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detectListeria monocytogenesin artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. ForListeriaspecies the measuring time was maximum 34 h. The absence ofL. monocytogenescould reliably be proven by the redox potential measurement method, butListeria innocuaandBacillus subtiliscould not be differentiated fromL. monocytogeneson the basis of the redox curves. The presence ofL. monocytogeneshad to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g ofL. monocytogenesin a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection ofL. monocytogenesin food.

Download Full-text

Detection of Shiga Toxin-ProducingEscherichia coliSerotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in Raw-Milk Cheeses by Using Multiplex Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02089-10 ◽

2011 ◽

Vol 77 (6) ◽

pp. 2035-2041 ◽

Cited By ~ 60

Author(s):

Jordan Madic ◽

Noémie Vingadassalon ◽

Carine Peytavin de Garam ◽

Muriel Marault ◽

Flemming Scheutz ◽

...

Keyword(s):

Real Time ◽

Genetic Markers ◽

Shiga Toxin ◽

Real Time Pcr ◽

Raw Milk ◽

Immunomagnetic Separation ◽

Genetic Profile ◽

High Genetic Diversity ◽

E Coli ◽

Three Samples

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive forstx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28fliCalleles were highly prevalent and could not be used as reliable targets for screening. Combinations ofstx,eaevariants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and includedstx-wzxO26-eae-β1(4.8%; 19 samples),stx-wzxO103-eae-ε (1.3%; five samples),stx-ihp1O145-eae-γ1(0.8%; three samples), andstx-rfbEO157-eae-γ1(0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seveneaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Threestx-negative andeaeβ1-positiveE. coliO26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC fromstx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagicE. coli(EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA,katP, andespP, as well as genomic O islands 71 and 122. Except for one strain, they all contained thestx1variant only, which was reported to be less frequently associated with human cases thanstx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

Download Full-text

Environmental Monitoring of Mycobacterium bovis in Badger Feces and Badger Sett Soil by Real-Time PCR, as Confirmed by Immunofluorescence, Immunocapture, and Cultivation

Applied and Environmental Microbiology ◽

10.1128/aem.00978-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7471-7473 ◽

Cited By ~ 34

Author(s):

F. P. Sweeney ◽

O. Courtenay ◽

V. Hibberd ◽

R. G. Hewinson ◽

L. A. Reilly ◽

...

Keyword(s):

Environmental Monitoring ◽

Cell Viability ◽

Real Time ◽

Species Identification ◽

Mycobacterium Bovis ◽

Real Time Pcr ◽

Immunomagnetic Capture

ABSTRACT Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.

Download Full-text

An evaluation of immunomagnetic separation-real-time PCR (IMS-RTiPCR) combined assay for rapid and specific detection of Escherichia coli O157:H7 in raw milk and ground beef

Food Science and Technology ◽

10.1590/fst.15818 ◽

2019 ◽

Vol 39 (4) ◽

pp. 955-961

Author(s):

Birce MERCANOGLU TABAN ◽

Sait Aykut AYTAC

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Ground Beef ◽

Raw Milk ◽

Immunomagnetic Separation ◽

Escherichia Coli O157 ◽

Specific Detection

Download Full-text

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

International Journal of Microbiology ◽

10.1155/2021/6632036 ◽

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.

Download Full-text