Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

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Abstract

Objective

Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1 ^® (Qiagen) and the manual QIAamp ^® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/ hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi.

Results

Whereas EZ1 ^® (Qiagen) and QIAamp ^® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1 ^® on the five remaining pathogens. DNA extraction using the semi-automated EZ1 ^® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

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High Prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol

Bédis Dridi, Mireille Henry, Amel El Khéchine … (2009)

Background The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. Methodology/Principal Findings Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae) yielded a sequence similarity of 99–100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. Conclusions/Significance In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.

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Enteric protozoa in the developed world: a public health perspective.

Damien Stark, Bradley S Fletcher, John Harkness … (2012)

Several enteric protozoa cause severe morbidity and mortality in both humans and animals worldwide. In developed settings, enteric protozoa are often ignored as a cause of diarrheal illness due to better hygiene conditions, and as such, very little effort is used toward laboratory diagnosis. Although these protozoa contribute to the high burden of infectious diseases, estimates of their true prevalence are sometimes affected by the lack of sensitive diagnostic techniques to detect them in clinical and environmental specimens. Despite recent advances in the epidemiology, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist, requiring further research. There is evidence that climate-related changes will contribute to their burden due to displacement of ecosystems and human and animal populations, increases in atmospheric temperature, flooding and other environmental conditions suitable for transmission, and the need for the reuse of alternative water sources to meet growing population needs. This review discusses the common enteric protozoa from a public health perspective, highlighting their epidemiology, modes of transmission, prevention, and control. It also discusses the potential impact of climate changes on their epidemiology and the issues surrounding waterborne transmission and suggests a multidisciplinary approach to their prevention and control.

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Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

C. Schooling, E Chapey, M Rabodonirina … (2013)

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

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Author and article information

Contributors

Estelle Menu: menu.estelle1@gmail.com

Charles Mary: charles.mary@ap-hm.fr

Isabelle Toga: isabelle.toga@ap-hm.fr

Didier Raoult: didier.raoult@gmail.com

Stéphane Ranque: stephane.ranque@ap-hm.fr

Fadi Bittar:

ORCID: http://orcid.org/0000-0003-4052-344X

fadi.bittar@univ-amu.fr

Journal

Journal ID (nlm-ta): BMC Res Notes

Journal ID (iso-abbrev): BMC Res Notes

Title: BMC Research Notes

Publisher: BioMed Central (London )

ISSN (Electronic): 1756-0500

Publication date (Electronic): 27 March 2018

Publication date PMC-release: 27 March 2018

Publication date Collection: 2018

Volume: 11

Electronic Location Identifier: 206

Affiliations

[1 ]Aix Marseille Univ, IRD, APHM, MEPHI, IHU-Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France

[2 ]Aix Marseille Univ, IRD, APHM, VITROME, IHU-Méditerranée Infection, Marseille, France

Author information

Fadi Bittar http://orcid.org/0000-0003-4052-344X

Article

Publisher ID: 3300

DOI: 10.1186/s13104-018-3300-2

PMC ID: 5869780

PubMed ID: 29587846

SO-VID: 69881760-7b2a-42a0-a056-653459319fbc

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 5 February 2018

Date accepted : 20 March 2018

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ScienceOpen disciplines: Medicine

Keywords: enteric parasites,protozoa,microsporidia,qpcr,dna extraction

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ScienceOpen disciplines: Medicine

Keywords: enteric parasites, protozoa, microsporidia, qpcr, dna extraction

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Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

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Most cited references 11

High Prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol

Enteric protozoa in the developed world: a public health perspective.

Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

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Cited by 22

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