Update on molecular techniques for diagnostic testing of infectious disease

Sellon, Rance K.

doi:10.1016/s0195-5616(03)00023-8

Cited by 22 publications

(19 citation statements)

References 51 publications

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“…The basics of PCR and its varied formats have been reviewed extensively. 100,107,115,151 DNA extracted from FFPE tissues will typically have a size limit of 300 to 400 base pairs (bp), and attempts to amplify targets over 600 bp should be avoided. Various studies have shown that RNA extracted from FFPE samples is often less than 200 nucleotides long.…”

Section: Nucleic Acid Extractionmentioning

confidence: 99%

Beyond H&E

et al. 2013

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Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets. Keywords cancer, ISH, IHC, nucleic acid analysis, molecular pathology, PCR, virology Technical ConsiderationsTissue Handling, Fixation, and Embedding Fixation of tissues in 10% neutral buffered formalin, followed by paraffin embedding (FFPE), has long been the standard approach of tissue preparation for microscopic evaluation. The chemical principle of formalin fixation is based on the crosslinking of proteins. Formalin has been used extensively because it is inexpensive and excellently preserves morphological detail in histological sections. However, the relatively recent development and increasing use of both protein and nucleic acid detection methods have revealed some significant shortcomings associated with the formalin-based fixation process. The effects of formalin fixation on DNA are chemical modification, DNA trapping, and potentially DNA fragmentation. Chemical modification, considered the main effect, results from the direct cross-linking of proteins to DNA. 68 In addition, extensive protein-protein cross-linking and the formation of protein networks result in DNA trapping. The combination of these effects makes it much more difficult to extract DNA from FFPE tissue than from unfixed tissue, especially when methods used for extraction from fresh tissues are used on FFPE tissue without any modifications. 60,83 In addition to interfering with the ability to extract nucleic acid, formalin fixation also causes DNA fragmentation, especially when the formalin used is inadequately buffered, which then leads to formic acid formation during the fixation process. This, in turn, results in depurination of the DNA, which then becomes susceptible to cleavage by hydroxyl ions and results in the formation of single-stranded breaks.60 Similar to DNA, RNA is also degraded and chemically modified as the result of formalin fixation. The most important chemical effect of formalin on RNA is the formation of monomethylol groups with purine bases. 77 This has a significant effect on reverse transcription ...

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Section: Nucleic Acid Extractionmentioning

confidence: 99%

Beyond H&E

et al. 2013

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“…Clients, who bear the financial burden, should be counseled as to the benefit and shortfalls of this testing before ordering molecular-based tests. An excellent review of molecular-based techniques for diagnostic testing of infectious diseases has appeared in a previous issue in this series [46].…”

Section: Molecular-based Methodsmentioning

confidence: 99%

Diagnostic Investigation of Emerging Viruses of Companion Animals

Kapil

Yeary

Johnson

2008

Veterinary Clinics of North America: Small Animal Practice

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In this article, the authors are specifically concerned with the timely and accurate detection of emerging diseases of small animals that are viral in origin. Veterinarians are bound to encounter emerging viruses in their practice. The problem is unavoidable, because viruses are highly mutagenic. Even the immune response dictates the nature of virus that evolves in a host. If the clinical signs and diagnostic methods fail to correlate, the veterinarian should work with the diagnostic laboratory to solve the diagnostic puzzle.

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“…This last reason can be a problem with viruses that undergo significant strain variation. In this case, primers should target highly conserved regions ideally [18]. For example, this has been an impediment to development of PCR assays that consistently work with all strains of FIV.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…For example, this has been an impediment to development of PCR assays that consistently work with all strains of FIV. RNA templates require reverse transcription before amplification; this step can be inefficient and may lead to false negative results [18].…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Methodology in diagnostic virology

Kennedy

2005

Veterinary Clinics of North America: Exotic Animal Practice

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Selection of proper assays and appropriate interpretation of results can be a challenge to the veterinary clinician. Assays vary in methodology, sensitivity, and specificity. These assays can be invaluable in attaining the correct diagnosis, but a clear understanding of the assay and the results is essential. To this end, communication with the laboratory personnel is crucial. Optimal sample selection, shipping recommendations, assay selection, and interpretation should be discussed with the laboratory staff.

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Update on molecular techniques for diagnostic testing of infectious disease

Cited by 22 publications

References 51 publications

Beyond H&E

Beyond H&E

Diagnostic Investigation of Emerging Viruses of Companion Animals

Methodology in diagnostic virology

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