运用基因芯片技术建立检测水产食品中常见病原微生物方法的研究

目的:利用基因芯片技术,以细菌16S rDNA和23S rDNA为靶序列筛选引物和探针,建立快速、准确的检测水产食品中肠道致病菌的方法。方法:将致病菌的16S rDNA和23S rDNA全序列进行软件比对,在可变区和恒定区分别设计特异性寡核苷酸探针和通用性引物,点样于玻片制成基因芯片。致病菌DNA经过通用引物扩增后与芯片上的探针杂交,然后通过扫描图像对结果进行判断。对基因芯片检测的灵敏度进行了评价,并对模拟污染样本进行了实际检测,以验证所建立的方法。结果:设计的4对通用引物在同一条件下能够扩增7种常见肠道致病菌。在均一的杂交条件下能够同时检测单核细胞增生利斯特菌、副溶血性弧菌、霍乱弧菌、金黄色葡萄球菌、弗氏志贺氏菌、鼠伤寒沙门氏菌和肠出血性大肠杆菌O157∶H7;以鼠伤寒沙门氏菌为对象,本方法的检测灵敏度可达到10~3 cfu/mL,实际检测模拟污染的样本的正确率达到100%。结论:建立的基因芯片系统可以准确而稳定地实现对7种水产食品中常见致病菌的通用检测,为食源性感染的诊治与预防提供了有效的技术手段和方法依据。     

Targeted Quantitation of Site-Specific Cysteine Oxidation in Endogenous Proteins Using a Differential Alkylation and Multiple Reaction Monitoring Mass Spectrometry Approach

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput.Oxidation of cysteine residues plays a critical role in modifying the structure and function of many proteins. Although cysteine oxidation is a tightly regulated biological process, nonenzymatic processes can contribute substantially to its levels, such as during oxidative stress. Regulatory oxidation states such as disulfide bonding and S-nitrosylation are readily modulated (1) and play an essential role in many physiological processes, including cell cycle, growth, death, and differentiation (2). In contrast, prolonged accumulation of reactive oxygen species is associated with many pathological conditions and leads to stable overoxidized states (sulfinic and sulfonic acid) that may disrupt redox regulation and protein function (3) and, in most cases, are thought to be nonregenerative.Assays capable of comprehensively assessing the dynamic changes in site-specific oxidation states are especially critical to understanding the contribution of redox status to many diseases. Numerous redox-sensitive proteins, including essential cellular regulators such as p53, have been described previously (for review, see ref. 4). However, technical factors have hampered the identification of specific site(s) of modification and characterization of their redox status in cells. Site-directed mutagenesis is often employed to determine whether specific cysteines have redox-regulated functional roles (1), but this approach provides no information on the oxidation status of the endogenous protein. In addition, cysteine oxidation is dynamically dependent on the concentration, location, and specificity of small-molecule oxidants (5) and regulators of various antioxidant enzymes (6). Thiol pKa (7), solvent accessibility, and subcellular compartment (8, 9) also contribute to the dynamics of cysteine oxidation. Because the interface between chronic oxidative stress and disruption of essential cellular signaling has substantial biological relevance to disease and age-related pathological conditions (10–13), there is a strong need to develop sensitive and flexible assays capable of quantifying dynamic changes in the redox status of specific endogenous proteins.Direct analysis of most regulatory cysteine modifications is not suitable for robust quantitation because the modifications tend to be labile and susceptible to artifactual changes. Accurate preservation of the thiol oxidation state is commonly achieved with a three-step differential alkylation labeling strategy in which nonoxidized cysteines are 1) labeled with a tag, 2) chemically reduced, and 3) labeled with a distinguishing tag. The value of this process is that it replaces the labile oxidation-modified cysteines with highly stable alkylated forms (1). Differential alkylation specifically targets cysteine oxidation species that are susceptible to reaction with chemical reductants (DTT or TCEP¹) because higher oxidation states such as sulfinic and sulfonic acid are chemically irreversible. Fluorescent or epitope tags have been employed to evaluate redox sensitivity at the protein level (14, 15); however, combining differential alkylation using stable isotope-labeled reagents with mass spectrometry simultaneously identifies the specifically oxidized site and quantifies its reversible oxidation status. Although labeled iodoacetic acid (16) and N-ethylmaleimide (17) (NEM) have often been used, commercial ICAT reagents have become the preferred stable isotope label for redox analysis (18–21). A recent ICAT-based study identified and quantified the reversible oxidation of 120 redox-sensitive cysteines in Escherichia coli in an unbiased manner (18). However, because of the limited sensitivity, dynamic range, number of testable conditions, and stochastic sampling inherent in unbiased proteomics experiments, this approach has limited utility for interrogating targeted moderate- to low-abundance proteins or comprehensively characterizing multiple cysteines within a single protein. The use of an ICAT-based approach for targeted analysis of specific proteins is significantly limited by both the difficulty in scaling down the ICAT protocol and the disruption of protein structure that occurs after alkylation with numerous ICAT adducts, each over a kilodalton in size, which may occlude antibody epitopes useful for immunoaffinity enrichment.To overcome these limitations, we developed a highly sensitive method, OxMRM, that integrates protein purification, differential alkylation using a generic d₅ stable isotope-labeled NEM, and multiple reaction monitoring (MRM). OxMRM can quantify the oxidation status, both reversible and irreversible, of virtually any targeted cysteine or protein, even if in low abundance. We validate the OxMRM approach using the low-abundance tumor suppressor protein p53 and an established overoxidizable signaling regulator, protein tyrosine phosphatase-1B (PTP1B) (22, 23). These proteins serve as benchmark examples of nuclear and cytoplasmic redox-regulated proteins bearing reversibly and irreversibly oxidized cysteines. The flexibility of the OxMRM method allows it to be applied to essentially any protein or cysteine of interest with equal ease, allowing in-depth, site-specific analysis of putative and established redox-sensitive proteins. This makes OxMRM an ideal complement to large-scale redox proteomics studies as a verification tool with high sensitivity, precision, accuracy, and capacity to assess numerous oxidation conditions.     

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission election microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.     

A Rapid and Simple Method for the Determination of Picogram Levels of 3-Methoxytyramine in Brain Tissue Using Liquid Chromatography with Electrochemical Detection

Abstract: A rapid and simple technique using solvent extraction, ion-pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3-methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3-methoxytyramine added to the samples is 45–50%. 3-Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3-methoxytyramine levels in rat striata, while catechol- O -methyltransferase inhibition with 3',4'-dihydroxy-2 methylpropiophenone completely depleted 3-methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D-amphetamine were also examined. The 3-methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.     

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed ^1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris ^3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins ^5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation ⁷ and pre-digestion with trypsin, which makes it difficult for clinical use. To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes ^8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner ^8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins ^8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.     

Quantitative,Label‐Free Proteomics in the Symptomatic Niemann–Pick,Type C1 Mouse Model Using Standard Flow Liquid Chromatography and Thermal Focusing Electrospray Ionization

Niemann–Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system. This abnormal accumulation results in a cascade of pathophysiological events including progressive, cerebellar neurodegeneration, among others. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive neurodegeneration remain unclear. In the current study, a) the use of a commercial, highly efficient standard flow‐ESI platform for protein biomarker identification is implemented and b) protein biomarkers are identified and evaluated at a terminal time point in the NPC1 null mouse model. In this study, alterations are observed in proteins related to fatty acid homeostasis, calcium binding and regulation, lysosomal regulation, and inositol biosynthesis and metabolism, as well as signaling by Rho family GTPases. New observations from this study include altered expression of Pcp2 and Limp2 in Npc1 mutant mice relative to control, with Pcp2 exhibiting multiple isoforms and specific to the cerebella. This study provides valuable insight into pathways altered in the late‐stage pathophysiology of NPC1.     

Determination of Thymidine Phosphorylase Activity in Human Blood Cells and Fibroblasts by a Nonradiochemical Assay Using Reversed-Phase High-Performance Liquid Chromatography

In this study, we demonstrated that the highest activity of thymidine phosphorylase (TP) was found in peripheral blood mononuclear (PBM) cells followed by that of thrombocytes and granulocytes whereas no activity of TP could be detected in erythrocytes. The activity of TP in leukocytes proved to be intermediate compared to the TP activity observed in PBM cells and granulocytes. The activity of TP also was readily detectable in human fibroblasts.     

Inhibition of Catechol-O-Methyltransferase by 6,7-Dihydroxy-3,4-Dihydroisoquinolines Related to Dopamine: Demonstration Using Liquid Chromatography and a Novel Substrate for O-Methylation

We report that 6,7-dihydroxy-3,4-dihydroisoquinolines related to dopamine are potent inhibitors of catechol-O-methyltransferase (COMT), but are not apparent substrates for the enzyme in vitro or in vivo. Three dihydroxy (catecholic) dihydroisoquinolines, including the 1-benzyl (DesDHP) and the 1-methyl (DSAL) analogs, were found to inhibit COMT activity in rat liver supernatant more effectively than the well-known inhibitor, tropolone. Inhibition of O-methylation was uncompetitive with substrate, and O-methylated products of the catecholic dihydroisoquinolines were undetectable. For these in vitro studies, a facile liquid chromatographic assay was developed utilizing as a site-specific substrate, 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline-1-carboxylate (salsolinol-1-carboxylate). This catechol produces only one phenolic product isomer when incubated with liver supernatant and S-adenosylmethionine. Following central injection of DSAL in rats, inhibition of brain COMT in vivo was indicated by the reduced brain levels of homovanillic acid, but not of 3,4-dihydroxyphenylacetic acid. Furthermore, O-methylated DSAL metabolites could not be detected in brain by liquid or gas chromatography. We suggest that 6,7-dihydroxy-dihydroisoquinolines are "nonmethylatable" COMT inhibitors because they exist as quinoidal tautomers resembling pyridones or tropolones rather than as catechols. Quinoid formation is supported by the fluorescence and ultraviolet spectra for DSAL and its O-methyl derivatives. The experiments reveal a new class of COMT inhibitors that may be of pharmacological and mechanistic value. Additionally, 3,4-dihydroisoquinolines could arise endogenously via oxidation of the 1,2,3,4-tetrahydroisoquinolines which are ingested or produced from cellular catecholamine condensations. However, it is unlikely that dihydroisoquinoline (e.g., DSAL) concentrations necessary to inhibit COMT significantly would be attained via endogenous pathways.     

Trace-Level Determination of Oxolinic Acid and Flumequine in Soil,River Bed Sediment,and River Water Using Microwave-Assisted Extraction and High-Performance Liquid Chromatography with Fluorimetric Detection

This work presents the optimization of analytical procedures for the determination of two antibiotics, oxolinic acid (OA) and flumequine (FL), in bed sediment, river water, and soil samples. Three extraction methods (microwave-assisted extraction (MAE), ultrasonication, and reflux) were tested, and the highest recoveries were obtained with MAE (94 ± 3% and 95 ± 3% for OA and FL, respectively). A solid-phase extraction (SPE) clean-up step was optimized by comparing two polymeric sorbents: Oasis HLB and Oasis MAX. The final extracts were analyzed by liquid chromatography with fluorimetric detection. Limits of detection (LOD) obtained for OA and FL in soil and sediment ranged from 0.3 to 0.5 µg kg^?1. Meanwhile, a novel SPE procedure was also implemented for OA and FL determination in river water samples. It also relied on the use of Oasis MAX, and recovery rates were in the range 90–94%; LODs were 2 ng L^?1 for both OA and FL. These methods were applied for the analysis of samples taken in the Seine River basin (France). The obtained results demonstrated the widespread occurrence of OA and FL, at ng L^?1 and µg kg^?1 levels in water and sediment/soil, respectively, and their persistence in the environment.     

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay

Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch^® vs. our microfluidic filter method revealed moderate correlation (R² = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.     

Discovery of a 29-Gene Panel in Peripheral Blood Mononuclear Cells for the Detection of Colorectal Cancer and Adenomas Using High Throughput Real-Time PCR

Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.     

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy,and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.     

Evaluation of Tetrahydropalmatine Enantiomers on the Activity of Five Cytochrome P450 Isozymes in Rats Using a Liquid Chromatography / Mass Spectrometric Method and a Cocktail Approach

The aim was to evaluate the effects of tetrahydropalmatine (THP) enantiomers on the activity of five cytochrome P450 (CYP450) isozymes in vivo. A liquid chromatography / mass spectrometric (LC‐MS) method was developed for simultaneous determination of five specific probe substrates including metoprolol (2D6), caffeine (1A2), dapsone (3A4), chlorzoxazone (2E1), and tolbutamide (2C9) in rat plasma. Analytes were separated with the mobile phase consisting of 0.1% acetic acid aqueous solution and acetonitrile in a gradient elution. The mass spectrometric detection via selected ion monitoring (SIM) was operated in both positive ion mode (for metoprolol m/z 268, caffeine m/z 195, and dapsone m/z 249) and negative ion mode (for chlorzoxazone m/z 168 and tolbutamide m/z 269) in the same run. Linear correlation was obtained (r² > 0.99) over the concentration range of 0.050–25.0 µg/mL for caffeine and dapsone, 0.025–10.0 µg/mL for metoprolol, 0.050–50.0 µg/mL for chlorzoxazone, and 0.25–100.0 µg/mL for tolbutamide. Intra‐ and interday precision were less than 12.09%. The matrix effect ranged from 87.50% to 109.25% and the absolute recoveries were greater than 70%. The method was successfully applied to evaluate the effect of THP enantiomers on the activity of CYP450 isozymes by a cocktail approach. The pharmacokinetic results of five probe drugs indicated that there were stereoselective differences between the two THP enantiomers, i.e., d‐THP had the potential to inhibit the activities of CYP2D6 and CYP1A2 isozymes, while l‐THP inhibited CYP1A2 isozyme and induced CYP3A4 and CYP2C9 isozymes. Chirality 27:551–556, 2015. © 2015 Wiley Periodicals, Inc.     

A Simple Quantitative Approach for the Determination of Long and Medium Chain Lipids in Bio-relevant Matrices by High Performance Liquid Chromatography with Refractive Index Detection

There is increasing attention in the literature towards understanding the behaviour of lipid-based drug formulations under digestion conditions using in vitro and in vivo methods. This necessitates a convenient method for quantitation of lipids and lipid digestion products. In this study, a simple and accessible method for the separation and quantitative determination of typical formulation and digested lipids using high performance liquid chromatography coupled to refractive index detection (HPLC–RI) is described. Long and medium chain lipids were separated and quantified in a biological matrix (gastrointestinal content) without derivatisation using HPLC–RI on C₁₈ and C₈ columns, respectively. The intra- and inter-assay accuracy was between 92% and 106%, and the assays were precise to within a coefficient of variation of less than 10% over the range of 0.1–2 mg/mL for both long and medium chain lipids. This method is also shown to be suitable for quantifying the lipolysis products collected from the gastrointestinal tract in the course of in vivo lipid digestion studies.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.