scholarly journals Detection of Mycobacterium paratuberculosis in raw cow’s milk using polymerase chain reaction (PCR) technique

2020 ◽  
Vol 34 (1) ◽  
pp. 83-86 ◽  
Author(s):  
Ihsan M. Ahmed ◽  
Raad A. Al-Sanjary ◽  
Haiffa H. Alkazaly
2018 ◽  
Vol 87 (2) ◽  
pp. 189-195
Author(s):  
Milena Alicja Stachelska

Lactococcus lactis subsp. cremoris belongs to lactic acid bacteria that play a crucial role in cheese production and it is known to be beneficial to human health. The aim of the study was to establish a rapid and accurate quantitative real-time polymerase chain reaction (qPCR) method to detect and enumerate L. lactis subsp. cremoris in artisanal raw cow’s milk cheese. Artisanal raw cow’s milk cheese samples were used to check for presence and number of L. lactis subsp. cremoris strains. The method applies a set of target-specific PCR (polymerase chain reaction) primers and a fluorogenic probe, and amplifies a part of the LACR_RS01280 gene that encodes the aminoacetone oxidase family flavin adenine dinucleotide (FAD) binding enzyme. All 5 L. lactis subsp. cremoris strains examined were found to be qPCR positive. There was no signal recorded for 8 strains which belong to closely related species. The limit of detection amounted to ten copies per reaction and the assay indicated a linear dynamic range of seven logs. This method may be applied in detection and enumeration of L. lactis subsp. cremoris in cheese during its ripening. Moreover, it may be applied to examine the distribution of L. lactis subsp. cremoris during the cheese production and ripening.


2002 ◽  
Vol 65 (2) ◽  
pp. 362-366 ◽  
Author(s):  
M. T. BOTTERO ◽  
T. CIVERA ◽  
A. ANASTASIO ◽  
R. M. TURI ◽  
S. ROSATI

A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled “pure buffalo” mozzarella.


2018 ◽  
Vol 87 (3) ◽  
pp. 301-308
Author(s):  
Milena Alicja Stachelska ◽  
Roberta Foligni

The first objective of this work included the development of real-time polymerase chain reaction (RT-PCR) which is also known as quantitative polymerase chain reaction (qPCR) assays to quantify two species of lactic acid bacteria which play a very important role in cheese ripening: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The second objective was the comparison of qPCR and plate counts of these two species present in raw cow’s milk cheese samples during different stages of ripening. Thirty-three deoxyribonucleic acid (DNA) samples coming from seven different bacterial species, which were phylogenetically related or commonly isolated from raw milk and dairy products, were chosen as positive and negative controls. The qPCR assays showed a high quantification capacity characterised by their linearity (R2 > 0.998), PCR efficiencies which were within the range 78.0–90.0% for L. delbrueckii subsp. bulgaricus, and 93.6–100.5% for S. thermophilus, and quantification limit (103 gene copies/ml for L. delbrueckii subsp. bulgaricus and 10 gene copies/ml for S. thermophilus). The importance of our study is in the monitoring of changes in populations of L. delbrueckii subsp. bulgaricus and S. thermophilus contributing to cheese ripening using the newly designed qPCR assay.


Gut ◽  
1997 ◽  
Vol 41 (5) ◽  
pp. 646-650 ◽  
Author(s):  
M P Riggio ◽  
J Gibson ◽  
A Lennon ◽  
D Wray ◽  
D G MacDonald

Background—Although intestinal Crohn’s disease has long been suspected to have a mycobacterial cause, possible mycobacterial involvement in orofacial granulomatosis (OFG) and oral lesions of Crohn’s disease has not yet been investigated.Aims—As the slow growingMycobacterium paratuberculosis has been implicated in the aetiology of intestinal Crohn’s disease, the potential involvement of this mycobacterial species in OFG and oral lesions of Crohn’s disease was investigated.Patients—To attempt detection of the organism in OFG and oral Crohn’s disease tissue samples, a polymerase chain reaction (PCR) assay was used on archival formalin fixed, paraffin wax embedded oral tissue sections from 30 patients with OFG, seven with Crohn’s disease, and 12 normal controls.Methods—The PCR assay used was based on primers targeting the 5′ region of the multicopy IS900 DNA insertion element of the M paratuberculosis genome. In order to achieve maximum sensitivity, two rounds of PCR were carried out and amplicons confirmed by Southern blot hybridisation to a digoxigenin labelled IS900 DNA probe.Results—None of the OFG and oral lesions of Crohn’s disease samples were positive forMparatuberculosis and all normal controls were also negative.Conclusions—These results suggest that M paratuberculosis may not be a major aetiological agent in OFG or oral Crohn’s disease lesions, although the use of paraffin wax embedded tissue as opposed to fresh tissue as a sample source could underestimate the true prevalence of the organism.


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