Mycobacterium avium Subspecies Paratuberculosis in the Inflamed Gut Tissues of Patients with Crohn’s Disease in China and its Potential Relationship to the Consumption of Cow’s Milk: A Preliminary Study

Cheng, Jun; Bull, Tim J.; Dalton, P.R.; Cen, S.; Finlayson, Caroline; Hermon‐Taylor, John

doi:10.1007/s11274-005-0809-z

Cited by 9 publications

(6 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We assessed the importance of host cell disruption (grinding and/or 1 vs. 2. vs. 3 bead beating steps) [3,4,26], the duration of lysozyme digestion (1h vs. 24h vs. 48h) [10,15,17], and the number of phenol:chloroform:isoamyl alcohol steps (1 vs. 2 washes) [3-6]. …”

Section: Methodsmentioning

confidence: 99%

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

et al. 2013

View full text Add to dashboard Cite

BackgroundNucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA.Principal FindingsRegarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89±0.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis).ConclusionsOur findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.

show abstract

Section: Methodsmentioning

confidence: 99%

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

et al. 2013

View full text Add to dashboard Cite

show abstract

“…As in paratuberculosis, mainly the terminal ileum and colon are affected and the disease is usually characterised by abdominal pain, general malaise, loss of energy and weight, and other non-specific symptoms [5,6]. A definitive explanation of CD causation does not yet exist, although MAP is thought to be one of the triggering factors [5][6][7][8][9][10]. Drinking water, water aerosol [11], milk [12][13][14][15] or even meat [16] are considered to be sources of MAP in human populations.…”

Section: Mycobacterium Avium Subsp Paratuberculosismentioning

confidence: 99%

Oligonucleotide Microarray Technology and its Application to Mycobacterium avium subsp. paratuberculosis Research: A Review

2009

View full text Add to dashboard Cite

Microarrays represent a modern powerful technology, which have potential applications in many areas of biological research and provide new insights into the genomics and transcriptomics of living systems. The aim of this review is to describe the application of microarray technology for Mycobacterium avium subsp. paratuberculosis (MAP) research. The main focus points include a summary of results obtained for MAP using microarrays, examination of the fields of MAP research which are currently being investigated and possible areas of future research. This article is divided into two parts according to the type of nucleic acid used for array hybridisation. Articles related to MAP research using microarray technology are then divided according to the field of study, such as comparative genome analysis, diagnostics, expression or environmental studies.

show abstract

“…Particularly, animals infected subclinically represent a hotspot for transmitting MAP within the herd [3]. In humans, MAP was isolated from patients with Crohn's disease (inflammatory bowel disease) [4–6] and for more than 100 years, MAP has been discussed to be the causative agent [7]. Johne's disease is leading to huge economic losses in dairy production and since no pharmacological treatment or a licensed vaccine are available, early detection of the source of a MAP infection in a herd is very crucial to diminish such losses.…”

Section: Introductionmentioning

confidence: 99%

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

et al. 2016

View full text Add to dashboard Cite

BackgroundThe detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.Methodology/Principal FindingsIn this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.Conclusions/SignificanceThe RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

show abstract

Mycobacterium avium Subspecies Paratuberculosis in the Inflamed Gut Tissues of Patients with Crohn’s Disease in China and its Potential Relationship to the Consumption of Cow’s Milk: A Preliminary Study

Cited by 9 publications

References 31 publications

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

Oligonucleotide Microarray Technology and its Application to Mycobacterium avium subsp. paratuberculosis Research: A Review

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

Contact Info

Product

Resources

About