Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica

Murphy, Niamh; McLauchlin, James; Ohai, C; Grant, Kathleen A.

doi:10.1016/j.ijfoodmicro.2007.06.006

Cited by 75 publications

(42 citation statements)

References 30 publications

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“…The amplification parameters were 95 uC for 5 min, followed by 95 uC for 15 s and 60 uC for 60 s. The cycle threshold was set at 0.05 for all targets except vtx1, for which it was set at 0.025. Faecal inhibition was monitored by adding an internal amplification control -the green fluorescent protein (gfp) gene (Murphy et al, 2007) -to the DNA extract. For all faecal specimens positive for vtx and/or eae (intimin), 10 colonies were picked from either the MacConkey or SMAC plate and retested by the same PCR.…”

Section: Methodsmentioning

confidence: 99%

Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli

Jenkins¹,

Lawson²,

Cheasty³

et al. 2012

Journal of Medical Microbiology

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The European Union Reference Laboratory (EU-RL) has produced guidelines for a real-time PCR for the detection of verocytotoxigenic Escherichia coli (VTEC). In this study, we validated the EU-RL assay on 545 strains of VTEC and evaluated the utility of the assay for the detection of VTEC from stool specimens. The validation study on cultures showed that the EU-RL VTEC PCR was 99.3% sensitive for the detection of vtx genes; only strains harbouring vtx2f genes were not detected. The assay was 100% sensitive and 100% specific for the detection of both the eae and O157 rfbE genes. In a prospective study involving 500 stool samples, the EU-RL VTEC PCR detected vtx genes in 12.4% of specimens, compared to 3.8% specimens found to be culturepositive for E. coli O157 using the Health Protection Agency national standard culture method. This study showed that the EU-RL VTEC assay was reliable and robust, and an effective rapid screening method for the detection of VTEC from stool specimens.

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Section: Methodsmentioning

confidence: 99%

Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli

Jenkins¹,

Lawson²,

Cheasty³

et al. 2012

Journal of Medical Microbiology

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“…In addition to the creation of a standard curve, the use of an internal amplification control (IAC) in RT-PCR is gaining acceptance (Hoofer et al 2003;Klerks et al 2004;Murphy et al 2007). A major concern when applying PCR for the detection of pathogens in environmental samples and foods is the reporting of false-negative results.…”

Section: Microarraymentioning

confidence: 99%

“…Inhibition of nucleic acid amplification during PCR can occur through the degradation and sequestration of target DNA and primers, a reduction in polymerase activity, or a number of other possible reasons (Wilson 1997). As a result, it is necessary to include a control strategy so that essential information is available to validate the PCR results (Murphy et al 2007). The IAC, which consists of a non-target DNA sequence from a known source, is included in the same reaction tube and is co-amplified with the target sequence (Hoofar et al 2003).…”

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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“…The need to include internal controls has been observed in various types of clinical samples, especially in materials in which purification of nucleic acids is difficult due to abundance of organic matter such as feces and food (41)(42)(43)(44)(45)(46). The inclusion in PCR assays of genes that constitute cells such as β-actin, β-globin, albumin and G3PD has been useful to evaluate the quality of samples in qPCR and PCR experiments (20,34,47,48).…”

Section: Discussionmentioning

confidence: 99%

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

Gonçalves¹,

Régis-da-Silva²,

Brito³

et al. 2012

J. Venom. Anim. Toxins incl. Trop. Dis

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Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes, G3PD2 was chosen for the formation of multiplex PCR systems. The two multiplex PCR systems (mVL and mACL) were reproducible in all species evaluated. Results of test samples (sensitivity, specificity and efficiency) suggest its use in routine diagnosis, research activities in medicine and veterinary medicine. Additionally, the systems designed to detect the G3PD gene are capable of combining with other targets used for molecular diagnosis of infectious diseases. Concerning leishmaniasis, the multiplex PCR systems can be used in epidemiological studies for the detection of new and classic reservoirs, which may contribute to the reliability of results and development of actions to control the disease.

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Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica

Cited by 75 publications

References 30 publications

Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli

Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli

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

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

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