Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides

Ruiz-Fuentes, Jenny Laura; Díaz, Alexis Vladimir Pinilla; Entenza, Anayma Elena; Frión, Yahima; Suárez, Odelaisy; Tôrres, Pedro; Armas, Yaxsier de; Acosta, Lucrecia

doi:10.1016/j.ijmyco.2015.06.005

Cited by 13 publications

(17 citation statements)

References 15 publications

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“…To achieve good results, good sampling, sample processing and DNA extraction is essential [63][64][65][66]. The successful amplification of a target sequence depends largely on the amount and quality of the nucleic acids extracted from the biological samples [64].…”

Section: Leprae Dna Extractionmentioning

confidence: 99%

“…Procedures commonly used for the isolation of nucleic acids do not obtain good results for M. leprae DNA extraction [68,73,74] and pretreatment steps with: freezing/thawing [75], bead beater with pearls [27,35] proteinase K [8,30,66,76] minimum 16 hours incubation [33], tris-HCL or tris-EDTA [44], detergents such as triton X-100 [22,77], and SDS [8,66,[76][77][78], alone or in combination should be included.…”

Section: Leprae Dna Extractionmentioning

confidence: 99%

“…Additionally, DNA extraction with phenol-chloroform mixture [66,79] can be used to obtain good quality DNA although it has several steps of sample handling, that can reduce performance and increase cross contamination [80]. Several studies have validated the ability of commercial kits: QIAamp DNA Mini Kit and DNeasy Blood & Tissue Kit (QIAGEN Inc., Valencia, CA) for obtaining both high concentration [79] and good quality DNA with the highest purity value [66] even in samples with highly degraded [72] or scarce DNA [46;79;46].…”

Section: Leprae Dna Extractionmentioning

confidence: 99%

“…Ear lobe and/or lesion lymph: Amplification of M. leprae DNA in lymph and slit skin smears has been reported and shown to have advantage over conventional methods [8,60,66]. The DNA extraction using these samples can increase both the sensitivity and specificity of the diagnosis and it can also be applied on stained slit skin smears [27,50,61,76] and on a FTA ® elute card (patented Whatman FTA technology) (Cat no.…”

Section: Suitable Samples For M Leprae Nucleic Acid Detectionmentioning

confidence: 99%

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Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia

Soto¹,

Muñoz²

2015

Mol Biol

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Leprosy is a chronic infectious disease caused by an obligatory intracellular mycobacteria Mycobacterium leprae, which presents tropism for Schwann cells and skin macrophages. Leprosy is a public health problem and early diagnosis is essential to avoid incapacities. The disease´s clinical presentation varies from few to widespread lesions and its diagnosis continues to be a challenge due to the low sensibility of the conventional methods, based on bacillary counts of skin smears and histopathology. Molecular techniques, especially the methods to identify M. leprae DNA based on polymerase chain reaction (PCR) have emerged as a support of the conventional methods for the analysis of clinical samples in difficult to diagnose cases, such as pure neural leprosy, indeterminate and paucibacillary leprosy. The technique has also proved useful in the study of leprosy transmission and monitoring résistance to the WHO recommended Multidrug treatment. Different biological samples can be analysed and there is no consensus in the molecular diagnostic techniques respect of the most efficient nucleic acid extraction method, most appropriate methodology and genetic target for PCR. These methods provide a very valuable option for confirmation of difficult clinical cases with scarce bacilli but requires a well-equipped laboratory and the high cost makes it inaccessible to be used as a routine diagnostic tool in most endemic countries.

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Section: Leprae Dna Extractionmentioning

confidence: 99%

Section: Leprae Dna Extractionmentioning

confidence: 99%

Section: Leprae Dna Extractionmentioning

confidence: 99%

Section: Suitable Samples For M Leprae Nucleic Acid Detectionmentioning

confidence: 99%

See 2 more Smart Citations

Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia

Soto¹,

Muñoz²

2015

Mol Biol

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“…These may not yet have been identified because they exhibit few indicative diagnostic characteristics through culture morphology (Kom arek et al 2014;Sciuto and Moro 2015), attenuated growth capacity or unclear growth requirements in culture, making them difficult to identify by traditional methodology (Youssef et al 2014;Sciuto and Moro 2015). In addition, they may not be able to be cultivated, thus known as viable, but not cultivable, which is either a transitory state for some bacteria in response to the environment stress, such as the Vibrio genus (Griffitt et al, 2011;Zhong et al 2016;Chahorm and Prakitchaiwattana 2018) or a stable state, as occurs in Mycobacterium leprae and Treponema pallidum (Petricha et al 2015;Ruiz-Fuentesa et al 2015;Gogarten et al 2016;Steinmann et al 2017). Thus, in both cases, applying molecular biology tools allows for their taxonomic classification, for the evaluation of their pathogenic potential and serve as a diagnostic method (Bergholz et al 2014;Das et al 2014;Kergourlay et al 2015;Sciuto and Moro 2015;King et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Application of molecular tools to elucidate the microbiota of seafood

2018

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The aim of this review is to present the methodologies currently applied to identify microbiota and pathogens transmitted to humans through seafood consumption, focusing on molecular techniques and pointing out their importance, advantages, disadvantages and applicability. Knowledge of available techniques allows researchers to identify which technique best fits their expectations. With such discernment, it will be possible to infer which disadvantages will be present and, therefore, not interfering with the final result. Two methodologies can be employed for this purpose, dependent and independent cultures. However, the dependent culture has certain limitations that can be solved through the independent cultivation techniques, such as PCR, PFGE and NGS, especially through the sequencing of the 16S rRNA region, providing a complete view of microbial diversity. These have revolutionized microbiological knowledge, mainly because they allow for the identification of uncultivable micro-organisms, which represent a substantial portion of total micro-organisms, making it possible to elucidate not yet described taxa which may display pathogenic potential, besides quantifying microbial communities, microbiota genetics, translated proteins and produced metabolites. In addition, transcriptomic and metabolomic techniques also allow for the evaluation of possible impacts that microbial communities may create in their environment, as well as the determination of potential pathogenicity to humans.

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Direct comparison of eDNA capture and extraction methods through measuring recovery of synthetic DNA cloned into living cells

Bockrath¹,

Tuttle‐Lau²,

Mize³

et al. 2022

Environmental DNA

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Before eDNA surveys can be deployed, the methods used to capture and extract DNA need to be determined. Multiple widely used eDNA capture and DNA extraction methods are available, and the effectiveness of these methods has been assessed in the literature. These studies used raw estimates of target cells captured from mesocosms and tanks, or already extracted DNA, which are not reflective of environmental DNA or precise enough to accurately compare eDNA capture and DNA extraction methods. Here, using qPCR quantification, we compared two eDNA capture and extraction methods using low and high concentrations of DNA cloned into living cells. By cloning target DNA into living cells, we can control the quantity of our starting copy number to directly compare the efficiency and effectiveness of each method throughout the process of eDNA handling and data analysis. This approach is useful for determining which capture and extraction method is suitable for the target species and sampling location of interest before full‐scale deployment. Although our target species DNA is consistently and accurately detected (even at low concentrations), stochasticity is a significant factor in our ability to consistently recover all the starting material by the end of sample processing. By recognizing that stochasticity is important for eDNA recovery, more accurate sampling models and eDNA processing protocols can be developed to increase eDNA detection sensitivity.

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Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides

Abstract: The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques.

Cited by 13 publications

References 15 publications

Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia

Leprosy Diagnosis: An Update on the Use of Molecular Tools Lucrecia

Application of molecular tools to elucidate the microbiota of seafood

Direct comparison of eDNA capture and extraction methods through measuring recovery of synthetic DNA cloned into living cells

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