Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions

Bickley, J.; Short, J.K.; McDowell, David G.; Parkes, Helen C.

doi:10.1111/j.1472-765x.1996.tb01131.x

Cited by 191 publications

(135 citation statements)

References 17 publications

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“…Milk and fecal samples are the most common specimens for paratuberculosis diagnosis. Milk is considered to be a difficult specimen for the detection of organisms by PCR, due to the presence of large amounts of fat and calcium ions (1,15). Fecal samples are often considered inappropriate for PCR due to the presence of large amounts of irrelevant genomic material and high concentrations of inhibitors such as bile salts, bilirubin, urobilinogens, and polysaccharides (13,32).…”

Section: Discussionmentioning

confidence: 99%

Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Khare¹,

Ficht²,

Santos³

et al. 2004

J Clin Microbiol

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Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and costeffective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.

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Section: Discussionmentioning

confidence: 99%

Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Khare¹,

Ficht²,

Santos³

et al. 2004

J Clin Microbiol

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“…Aliquots of 1 ml of these dilutions were centrifuged for 5 min at 7,000 ϫ g and submitted to four washes, and then the pellets were resuspended in 1 ml of 1ϫ PBS and centrifuged at 5,000 ϫ g for 5 min. PBS rather than water was used for washes to precipitate calcium ions from the milk, which is known to be a PCR inhibitor (8), and to protect the bacterial membranes from lysis during these steps. After the last centrifugation, the pellet was resuspended in 1 ml of autoclaved distilled water to allow the burst of bacterial cells.…”

Section: Methodsmentioning

confidence: 99%

Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR

et al. 2001

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Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 ؋ 10 2 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 ؋ 10 3 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.Bovine mastitis (BM) is an inflammation of the mammary gland, usually due to a microbial infection (28), which causes North American dairy producers to lose billions of dollars every year. These losses are primarily due to lower milk yields, reduced milk quality, and higher production costs. BM often becomes chronic, and it is important to identify quickly the new clinical cases in order to control infection in the herd. The bacteria responsible for BM can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis) or contagious (Staphylococcus aureus and Streptococcus agalactiae) depending of their primary reservoir (environment versus infected mammary gland quarter) (11,25).The suitability of a detection method for routine diagnosis depends on several factors, such as specificity, sensitivity, expense, amount of time, and applicability to large numbers of milk samples. The most common but unspecific method (2) to identify potential chronic infections is a somatic cell count: the California Mastitis Test in field conditions and the automated method in the diagnosis laboratory. Currently, the method of identification of the mammary gland pathogens is by in vitro culture, which provides the "gold standard"; however, this technique is labor-intensive and time-consuming. Two other problems can be encountered when these methods of identification are used: first, 2 to 3 days are required to grow, isolate, and identify the pathoge...

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“…However all mycoplasma species could be detected when mycoplasmas were collected from the simulated specimens by centrifugation and lysed with a mycoplasmal lysis buffer. Because a large number of milk components remain in template DNA in milk samples, these components may have interfered with the PCR reaction [2,16,18]. Therefore, we tried to eliminate milk components from the simulated specimens by centrifugation.…”

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confidence: 99%

Detection of Mycoplasma in Mastitic Milk by PCR Analysis and Culture Method.

Hirose

Kawasaki

Kotani

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. Mycoplasma alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis were directly detected from milk specimens by a polymerase chain reaction (PCR) when milk specimens were centrifuged and treated with mycoplasmal lysis buffer. The sensitivity of this PCR method was 110 to 1,400 colony forming units (CFU). This method was useful for the detection of mycoplasmas in milk specimens from cows at an early stage of mycoplasmal mastitis since a small amount of mycoplasma could be detect in milk without culture. The results were available within 12 hr, which is faster than conventional culture techniques. M. bovirhinis was detected in more than 70% of mastitic milk specimens when mycoplasmas were detected in milk specimens from 30 cows with mastitis by this PCR method. KEY WORDS: mastitis, mycoplasma, PCR.J. Vet. Med. Sci. 63(6): 691-693, 2001 Mycoplasma mycoides subsp. mycoides, M. bovis, M. alkalescens, M. bovigenitalium, M. dispar and others are the causative agents of several diseases in cows and calves, such as mastitis, pneumonia, arthritis, genital disorders and abortions [1,3,6,12,15]. M. bovirhinis is a troublesome agent as a secondary invader in respiratory diseases, and it is the mycoplasma that is the most common isolate from the nasal cavity of cattle with respiratory disease [8]. Since the diseases are largely resistant to chemotherapy [6,24], it is necessary to have rapid and reliable diagnostic methods to detect the agents at an early stage, so that effective control measures can be introduced in time. However, current methods for the detection of mycoplasmas are restricted to culture and serology, and both of these diagnostic methods are time consuming, laborious and difficult. Isolation and identification of mycoplasmas may take weeks, and few laboratories have the capabilities required to routinely culture mycoplasmas. Serodiagnosis requires the demonstration of increasing antibody titers that are reached only ten to fourteen days after the onset of clinical symptoms [19]. Consequently, the pathogen cannot be detected during the incubation period. Moreover, the serological cross reactions among the mycoplasma species are a critical problem [19]. Therefore, the absence of practical and rapid detection methods and resistance to therapy hamper the effective control of mycoplasma infections.Gonzalez [7] and Hatanaka et al. [9] reported cases of mycoplasmal mastitis caused by M. bovis derived from the nasal secretion of cows with pneumonia, emphasizing the relationship between mycoplasmal pneumonia and mastitis. During 1996-1997, we performed nationwide research on the isolation of mycoplasma from the nasal secretions of calves with respiratory diseases, and the result was that a strain of mycoplasma was isolated from about 30% of calves with respiratory diseases. The majority of the isolates consisted of the following 4 species, M. alkalescens, M. bovigenitalium, M. bovirhinis and M. bovis (Data not shown). Therefore, we restrict discussion to these four species in this study.The purpose of...

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Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions

Cited by 191 publications

References 17 publications

Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR

Detection of Mycoplasma in Mastitic Milk by PCR Analysis and Culture Method.

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