scholarly journals Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

2022 ◽  
Vol 8 ◽  
Author(s):  
Raul Leal Faria Luiz ◽  
Rodrigo Caldas Menezes ◽  
Sandro Antonio Pereira ◽  
Raquel de Vasconcellos Carvalhaes de Oliveira ◽  
Manoel Marques Evangelista Oliveira
Keyword(s):  
Dna Extraction ◽  
Nested Pcr ◽  
Paraffin Section ◽  
Chemical Extraction ◽  
Dimorphic Fungi ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Formalin Fixed ◽  
Skin Samples ◽  
Positive Controls

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genetic Variants ◽  
Variant Calling ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Control Samples

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

2011 ◽  
Vol 64 (3) ◽  
pp. 252-256 ◽  
Author(s):  
Gernot Kriegshäuser ◽  
Veronika Auner ◽  
Eva Schuster ◽  
Barbara Holzer ◽  
Christian Oberkanins ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Ovarian Tissue ◽  
Analytical Sensitivity ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Sensitivity Specificity ◽  
Formalin Fixed ◽  
Reverse Hybridisation Assay

AimsTo evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.MethodsThe strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.ResultsThe strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.ConclusionsThe strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.

scholarly journals Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues

2020 ◽  
Author(s):  
Joseph Jillwin ◽  
Shivaprakash M. Rudramurthy ◽  
Shreya Singh ◽  
Amanjit Bal ◽  
Ashim Das ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Molecular Identification ◽  
Nested Pcr ◽  
18S Rdna ◽  
Test Accuracy ◽  
Fungal Identification ◽  
Ffpe Tissues ◽  
Fungal Dna ◽  
Pcr Targeting ◽  
Formalin Fixed

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level Gap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation. Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues. Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae. Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues. Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

scholarly journals Superiority of Formalin-Fixed Paraffin-Embedded Brain Tissue for in vitro Assessment of Progressive Supranuclear Palsy Tau Pathology With [18F]PI-2620

2021 ◽  
Vol 12 ◽  
Author(s):  
Marie Willroider ◽  
Sigrun Roeber ◽  
Anja K. E. Horn ◽  
Thomas Arzberger ◽  
Maximilian Scheifele ◽  
...  
Keyword(s):  
Progressive Supranuclear Palsy ◽  
Brain Tissue ◽  
Tau Pathology ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Frozen Tissue ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Disease Entities ◽  
Formalin Fixed

Objectives: Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of autoradiography signal using the novel next-generation tau positron emission tomography (PET) radiotracer [18F]PI-2620 with immunohistochemically determined tau-protein load in both formalin-fixed paraffin-embedded (FFPE) and frozen tissue samples of patients with Alzheimer's disease (AD) and Progressive Supranuclear Palsy (PSP).Methods: We applied [18F]PI-2620 autoradiography to postmortem cortical brain samples of six patients with AD, five patients with PSP and five healthy controls, respectively. Binding intensity was compared between both tissue types and different disease entities. Autoradiography signal quantification (CWMR = cortex to white matter ratio) was correlated with the immunohistochemically assessed tau load (AT8-staining, %-area) for FFPE and frozen tissue samples in the different disease entities.Results: In AD tissue, relative cortical tracer binding was higher in frozen samples when compared to FFPE samples (CWMRfrozen vs. CWMRFFPE: 2.5-fold, p &lt; 0.001), whereas the opposite was observed in PSP tissue (CWMRfrozen vs. CWMRFFPE: 0.8-fold, p = 0.004). In FFPE samples, [18F]PI-2620 autoradiography tracer binding and immunohistochemical tau load correlated significantly for both PSP (R = 0.641, p &lt; 0.001) and AD tissue (R = 0.435, p = 0.016), indicating a high agreement of relative tracer binding with underlying pathology. In frozen tissue, the correlation between autoradiography and immunohistochemistry was only present in AD (R = 0.417, p = 0.014) but not in PSP tissue (R = −0.115, p = n.s.).Conclusion: Our head-to-head comparison indicates that FFPE samples show superiority over frozen samples for autoradiography assessment of PSP tau pathology by [18F]PI-2620. The [18F]PI-2620 autoradiography signal in FFPE samples reflects AT8 positive tau in samples of both PSP and AD patients.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genetic Variants ◽  
Variant Calling ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Control Samples

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Variant Calling ◽  
Extraction Methods ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Dna Extraction Methods

Abstract Background: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92 % of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Canine pyoderma: mecA persists autogenous bacterin formulation from meticillin-resistant Staphylococcus pseudintermedius (MRSP) and S. aureus (MRSA)

2021 ◽  
Vol 49 (06) ◽  
pp. 437-440
Author(s):  
Anette Loeffler ◽  
Claire Scott ◽  
Siân-Marie Frosini
Keyword(s):  
Dna Extraction ◽  
Clinical Isolates ◽  
Fixed Tissue ◽  
Staphylococcus Pseudintermedius ◽  
Tissue Samples ◽  
Formalin Fixed Tissue ◽  
Naked Dna ◽  
Skin Bacteria ◽  
Formalin Fixed

Abstract Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

scholarly journals Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

2012 ◽  
Vol 32 (8) ◽  
pp. 715-720 ◽  
Author(s):  
Paula R. Almeida ◽  
Caroline P. Andrade ◽  
Laura L. Almeida ◽  
Luiz G.S. Oliveira ◽  
Luiza A. Castro ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Mycoplasma Hyopneumoniae ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Histological Features ◽  
Frozen Tissue ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Optimization of genomic DNA extraction from formalin-fixed fish specimens

2013 ◽  
Vol 19 (6) ◽  
pp. 1068-1073
Author(s):  
Xiaolan KONG ◽  
Zuozhi CHEN ◽  
Lin LIN ◽  
Chunhou LI ◽  
Peiwen LIANG
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Genomic Dna Extraction ◽  
Formalin Fixed
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