Quantitative Detection of
            <i>Clostridium tyrobutyricum</i>
            in Milk by Real-Time PCR

López-Enríquez, Lorena; Rodrı́guez-Lázaro, David; Hernández, Marta

doi:10.1128/aem.02642-06

Cited by 54 publications

(37 citation statements)

References 23 publications

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“…Abbatecola et al (1) and Retief et al (29) used nested PCR to detect P. chlamydospora in grapevine rootstocks and in nurseries. However, real-time PCR proved more sensitive, and its results were more reproducible for these species (12) and also for others (21). The advantages of real-time PCR detection over conventional methods are its specificity, sensitivity, and speed.…”

Section: Discussionmentioning

confidence: 99%

Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Martín

Cobos

Martin

et al. 2012

Appl Environ Microbiol

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ABSTRACTPhaeomoniella chlamydosporaandPhaeoacremonium aleophilumare the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification ofP. chlamydosporaandP. aleophilum. Specificity was demonstrated against purified DNA from 60P. chlamydosporaisolates or 61P. aleophilumisolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg forP. chlamydosporaand 50 fg forP. aleophilum. Detection was specific and sensitive forP. chlamydosporaandP. aleophilum. Spores ofP. chlamydosporaandP. aleophilumwere detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification.P. chlamydosporawas detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.

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Section: Discussionmentioning

confidence: 99%

Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Martín

Cobos

Martin

et al. 2012

Appl Environ Microbiol

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“…Although sample enrichment does increase sensitivity, it essentially renders RT-PCR non-quantitative. RT-PCR has also been used to quantify ammonia-oxidizing bacteria in soil (Hermansson and Lindgren 2001), enterococci and human adenovirus in water (He and Jiang 2005), Vibrio vulnificus in shellfish and water (Panicker et al 2004), Lactobacillus salivarius in broiler chickens (Harrow et al 2007) and Clostridium tyrobutyricum in milk (López-Enríquez et al 2007).…”

Section: Microarraymentioning

confidence: 99%

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

Dungan

Leytem

2009

World J Microbiol Biotechnol

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The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs) is a concern from a human and animal health perspective. To better understand the airborne microorganisms found in these environments, a number of collection and analytical techniques have been utilized and will be discussed in this review. The most commonly used bioaerosol collection method is the liquid impingement format, which is suitable with a number of culture-based and non-culture molecularbased approaches, such as polymerase chain reaction. However, the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses. Because of the limitations often associated with culturebased analyses, we focused our discussion on the application of molecular-based techniques to identify and/or quantify microorganisms, as they have promising application in bioaerosol research. The ability to rapidly characterize airborne microorganisms will help to ensure protection of public and environmental health.

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“…In three independently replicated experiments, 1-ml portions of phosphate-buffered saline containing approximately 2.5 ϫ 10 7 , 2.5 ϫ 10 6 , 2.5 ϫ 10 5 , 2.5 ϫ 10 4 , 2.5 ϫ 10 3 , 250, 25, or 3 M. agalactiae cells were added to 50-ml centrifuge tubes, each containing 25 ml of raw sheep milk. DNA was purified from milk by the procedure described by López-Enríquez and coworkers (14), with the only modification being homogenization for 10 min in a homogenizer (Pulsifier) prior to DNA extraction. We consistently (Table 3).…”

Section: Capri M Mycoides Subsp Mycoides Lc and M Putrefaciens)mentioning

confidence: 99%

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples

et al. 2009

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We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R 2 > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.

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Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR

Cited by 54 publications

References 23 publications

Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples

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