Comparison of Five Commercial Nucleic Acid Extraction Kits for the PCR-based Detection of Burkholderia Pseudomallei DNA in Formalin-Fixed, Paraffin-Embedded Tissues

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Abstract

The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR.

FFPE samples from organs of Burkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targeting B. pseudomallei-specific sequences of varying length after 16 years of storage.

The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal.

The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits.

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Most cited references 17

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Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses.

Natalie Ludyga, Barbara Grünwald, Omid Azimzadeh … (2012)

Tissues used for clinical diagnostics are mostly formalin-fixed and paraffin-embedded (FFPE) which provides many advantages. However, the quality of the obtained nucleic acids (NA) is reduced and this turns out to be a challenge for further molecular analyses. Although the spectrum of analyses of NA extracted from FFPE tissue has increased, the standard operating procedures for NA isolation from old tissue blocks still need to be improved. Here, we compared the efficiency of different NA extraction methods, using FFPE tissues of variable age and origin, with respect to downstream analyses. Our study showed that the phenol-chloroform isoamyl alcohol (PCI) and the commercial Qiagen protocol yielded samples with highest purity. The PCI protocol delivered the longest amplicons even from samples from the 1970s. We developed a short (1 h) tissue lysis procedure that turned out to be highly time- and cost-effective when DNA quality was tested using single and multiplex PCR. Compared to a 1-day lysis-protocol, the amplicons were only 100 bp shorter. In addition, single-copy genes used in daily routine were successfully amplified from long-term stored FFPE samples following 1-h tissue-lysis. The RNA integrity numbers (RIN) determined on RNA isolated from FFPE tissues indicated degraded RNA; however, all RINs were above the generally agreed threshold of 1.4. We showed that, depending on the purpose of the analysis, NA retrieved from FFPE tissues older than 40 years may be successfully used for molecular analysis.

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Quantitation of viral load using real-time amplification techniques.

H Niesters (2001)

Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA)-based systems combined with several detection strategies are being employed in a clinical diagnostic setting. The importance of these assays in disease management is still in an exploration phase. Although these technologies have the implicit capability of accurately measuring DNA and RNA in clinical samples, issues related to standardization and quality control must be resolved to enable routine implementation of these technologies in molecular diagnostics. Copyright 2001 Elsevier Science (USA).

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Management of melioidosis.

Zachary S. Peacock, Vanaporn Wuthiekanun (2006)

Melioidosis is a serious human infection caused by the environmental Gram-negative bacterium Burkholderia pseudomallei. Outcome following melioidosis remains poor despite 20 years of clinical research. Overall mortality is 50% in north-east Thailand (35% in children) and 19% in Australia. Relapse is common (13% over 10 years), and results from failure to eradicate the organism. Treatment is required to complete 12-20 weeks, or longer if clinically indicated. This is divided into intravenous and oral phases. Clinical trial evidence supports the use of ceftazidime or a carbapenem antibiotic for initial parenteral therapy, which should be administered for at least 10-14 days. This is followed by a prolonged course of oral antimicrobial therapy with trimethoprim-sulfamethoxazole (TMP-SMX) with or without doxycycline. Amoxicillin-clavulanate is an alternative for children, pregnant women and for patients with intolerance to first-line therapy. Resistance of B. pseudomallei to these drugs is rare, with the exception of TMP-SMX; resistance rates are approximately 2.5% in Australia and 13-16% in Thailand. There is a lack of evidence for the value of adjunctive therapies in the treatment of melioidosis. Future studies aim to address whether meropenem is superior to ceftazidime during parenteral therapy, and whether doxycycline is a necessary component of oral treatment.

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Author and article information

Journal

Journal ID (nlm-ta): Eur J Microbiol Immunol (Bp)

Journal ID (iso-abbrev): Eur J Microbiol Immunol (Bp)

Journal ID (publisher-id): EUJMI

Title: European Journal of Microbiology & Immunology

Publisher: Akadémiai Kiadó (Budapest )

ISSN (Print): 2062-509X

ISSN (Electronic): 2062-8633

Publication date (Electronic): 29 September 2016

Publication date Collection: 29 September 2016

Volume: 6

Issue: 3

Pages: 244-252

Affiliations

[1 ] Chemical Faculty, University of Hamburg , Hamburg, Germany

[2 ]Friedrich Loeffler Institute, Federal Research Institute for Animal Health , Jena, Germany

[3 ]NATO Center of Excellence for Military Medicine (MilMedCOE), Deployment Health Surveillance Capability (DHSC) , Munich, Germany

[4 ] Department of Tropical Medicine at the Bernhard Nocht Institute , German Armed Forces Hospital of Hamburg, Hamburg, Germany

[5 ]Institut for Medical Microbiology, Virology and Hygiene, University Medicine Rostock , Rostock, Germany

Author notes

* Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Bernhard Nocht street 74, 20359 Hamburg, Germany; 0049-40-6947-28700; 0049-40-6947-28709; Frickmann@ 123456bni-hamburg.de

⁺ These authors contributed equally to this work.

Declaration of interest

The authors declare that there are no conflicts of interest.

Article

DOI: 10.1556/1886.2016.00028

PMC ID: 5063018

SO-VID: 142c09e3-816e-4df4-98be-82ac583c69a4

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 01 August 2016

Date accepted : 26 August 2016

Page count

Figures: 0, Tables: 7, Equations: 0, References: 17, Pages: 9

Funding

Funding sources There has been no source of funding.

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