One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish

Kotłowski, Roman; Martin, Anandi; Ablordey, Anthony; Chemlal, Karim; Fonteyne, Pierre-Alain; Portaels, Françoise

doi:10.1099/jmm.0.45593-0

Cited by 75 publications

(87 citation statements)

References 35 publications

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“…This hypothesis maintains that M. ulcerans is found in biofi lms of aquatic habitats and concentrated by grazing or fi lter-feeding invertebrates that are then consumed by predators known to bite humans (11). Initial evidence for this hypothesis used PCR detection of the insertion sequence IS2404 to document M. ulcerans' association with biting water bugs (Hemiptera), fi ltered concentrates of water, detritus, and aquatic plants (4,(12)(13)(14). These studies were important for understanding the possible environmental reservoirs of M. ulcerans.…”

mentioning

confidence: 99%

Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease

Benbow

Williamson

Kimbirauskas

et al. 2008

Emerg. Infect. Dis.

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Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans and associated with exposure to aquatic habitats. To assess possible transmission of M. ulcerans by aquatic biting insects, we conducted a fi eld examination of biting water bugs (Hemiptera: Naucoridae, Belostomatidae, Nepidae) in 15 disease-endemic and 12 non-disease-endemic areas of Ghana, Africa. From collections of 22,832 invertebrates, we compared composition, abundance, and associated M. ulcerans positivity among sites. Biting hemipterans were rare and represented a small percentage (usually <2%) of invertebrate communities. No signifi cant differences were found in hemipteran abundance or pathogen positivity between disease-endemic and nondisease-endemic sites, and between abundance of biting hemipterans and M. ulcerans positivity. Therefore, although infection through insect bites is possible, little fi eld evidence supports the assumption that biting hemipterans are primary vectors of M. ulcerans. M ycobacterium ulcerans infection is an emerging skin disease often called Buruli ulcer (BU). Infection results in illness and lasting negative socioeconomic effects in rural areas of the tropics and subtropics (1). The pathologic changes, clinical signs and symptoms, and treatment have been reviewed elsewhere (2-5). In this article we evaluate fi eld evidence for the potential of aquatic invertebrates to be vectors of M. ulcerans.The exact mode of BU transmission remains unknown; however, past epidemiologic studies have associated BU with human activity near, or within, slow-fl owing or standing water bodies that have been created or disturbed by humans (2-4). Although several water-related risk factors have been recognized, none has been consistently reported, making it diffi cult to identify specifi c water-related risk activities (6-8). Most studies suggest that infection occurs through inoculation of M. ulcerans into skin lesions or insect bites (2,4,9-11). Portaels et al. (11) were the fi rst to propose that aquatic insects might serve as vectors of M. ulcerans. This hypothesis maintains that M. ulcerans is found in biofi lms of aquatic habitats and concentrated by grazing or fi lter-feeding invertebrates that are then consumed by predators known to bite humans (11). Initial evidence for this hypothesis used PCR detection of the insertion sequence IS2404 to document M. ulcerans' association with biting water bugs (Hemiptera), fi ltered concentrates of water, detritus, and aquatic plants (4,(12)(13)(14). These studies were important for understanding the possible environmental reservoirs of M. ulcerans. However, IS2404 is now understood to be not specifi c for M. ulcerans because this insertion sequence has been found in a number of other aquatic mycobacterial species, including M. marinum (15)(16)(17). When more discriminatory methods based on detection of variable number tandem repeats were used, many IS2404-positive environmental samples were reported to lack M. ulcerans (18). In light of these recent fi ndings, the relative fr...

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mentioning

confidence: 99%

Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease

Benbow

Williamson

Kimbirauskas

et al. 2008

Emerg. Infect. Dis.

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“…19 The guanidine lysis method has been used to extract mycobacterial DNA in human specimens. 14 The application of this method in Johne's diagnosis is limited due largely to its inefficiency in removing PCR inhibitors originating from bovine fecal specimens. 3 In the current study, additional procedures were incorporated into the traditional guanidine lysis method to make it more suitable for the purification of MAP DNA from bovine fecal samples.…”

Section: Discussionmentioning

confidence: 99%

“…20 The combination of mechanical disruption and 1-tube chemical lysis/extraction allowed for maximal recovery of MAP DNA and reduction of cross-contamination. 14 The chelex matrix absorbed metal ions that could catalyze DNA degradation and inhibit downstream PCR reaction. 15 Finally, a mini-column purification procedure quickly removed the contaminating carbohydrates.…”

Section: Discussionmentioning

confidence: 99%

“…2,8,10,13 To develop highly sensitive and specific molecular detection methods, research has been focused on finding target sequences that are specific to MAP and abundant for detection, choosing suitable equipments and optimal PCR and/or hybridization conditions, evaluating DNA extraction methods, and optimizing techniques for recovering MAP organisms from clinical specimens. 2,9,11,14,17 The insertion sequence IS900, a 1451 base-pair (bp) repetitive element present in 12-20 copies in MAP genome, is commonly targeted for the detection of MAP by PCR. 2,10 Other sequences, such as hspX and F57, have also served as amplification targets for MAP PCR.…”

Section: Introductionmentioning

confidence: 99%

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An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Zhang

2011

J VET Diagn Invest

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Abstract. Due to the lipid rich cell wall of Mycobacterium avium subspecies paratuberculosis (MAP), the complex nature of bovine feces, and intermittent organism shedding by infected cattle, it is difficult to recover a sufficient amount of high-quality MAP DNA from fecal samples, directly affecting the sensitivity of downstream polymerase chain reaction (PCR) tests. In the current study, a DNA extraction method, designated the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, was developed for PCR-based detection of MAP in bovine fecal samples. The MVRDL method combined multiple procedures, including chemical pretreatment, 1-tube cell lysis and extraction, chelex matrix absorption, and mini-column purification. The DNA yield and purity, as measured by spectrophotometry, was 3.36 fg per colony forming unit (CFU) MAP and A260/280 absorbance ratio of 2, respectively. This method was further evaluated by real-time PCR. A linear correlation was found between cycle-threshold (Ct) and log input CFU (ranging from 7.2 to 7.2 3 10 7 CFU per ml or CFU per g). The detection limit of the realtime PCR assay was 3 CFU per ml of MAP culture or per g of MAP-spiked feces. In addition, the MVRDL method was validated by performing 7 Johne's direct fecal PCR proficiency tests administered by the National Veterinary Service Laboratories. Based on culture results as the ''gold standard,'' the specificity of MVRDL PCR was 100%, and the sensitivity was 98.46% for samples containing more than 1.5 CFU per tube of fecal cultures. To the authors' knowledge, this is the most efficient MAP DNA extraction method in comparison with all previously published protocols.

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“…Aseptic necropsy also reduces chances of detecting extraneous, environmentally derived mycobacteria in fish tissues when using molecular techniques such as PCR. A variety of PCR assays have been utilized for specific and rapid detection of piscine mycobacteria (Knibb et al 1993, Talaat et al 1997, Bruno et al 1998, Kotlowski et al 2004.…”

Section: Reports Of Mycobacteriosis In Striped Bassmentioning

confidence: 99%

Comparative analysis of mycobacterial infections in wild striped bass Morone saxatilis from Chesapeake Bay

Kaattari¹,

Rhodes²,

Kator³

et al. 2005

Dis. Aquat. Org.

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During an ongoing epizootic of mycobacteriosis, wild striped bass Morone saxatilis from Chesapeake Bay were analyzed using 3 methods for detection of either mycobacterial infection or associated granulomatous pathology. The specific detection techniques, which utilized aseptically collected splenic tissue, were histology, quantitative culture and nested PCR. Based on analysis of 118 samples, detection of infection differed significantly between the 3 methods (chi-square, p = 0.0007). Quantitative culture and nested PCR detected similar, higher rates of infection (69 and 75%, respectively) than the histological method (52%). Although primary PCR assays for a 924 to 940 bp segment of the mycobacterial 16S rRNA gene were positive for genomic DNA from mycobacterial cultures, a secondary, nested PCR reaction for an internal 300 bp gene segment was required in order to detect mycobacteria within splenic tissue. A similar rate of mycobacterial infection was present in fish collected from all sites tested. Although all detection methods found that striped bass age 4.0 to 4.9 yr had the highest positive incidence, nested PCR detected a higher frequency of mycobacterial infection in fish ≥ 6.0 yr of age than the other 2 methods. Quantitative bacteriology was a more sensitive detection technique when the fish tissue contained ≤10 3 mycobacteria g -1 .

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One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish

Cited by 75 publications

References 35 publications

Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease

Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Comparative analysis of mycobacterial infections in wild striped bass Morone saxatilis from Chesapeake Bay

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