DNA Probe Assay Research Articles

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

Blastomycosis: An Underrecognized Climate Sensitive Fungal Pathogen Causing Pneumonia in 52-year-old Immunocompetent Women from Southwestern Ohio—A Case Report and Literature Review

Blastomycosis, formerly known as Gilchrist disease, Chicago disease, North American blastomycosis, or Namekagos fever, is an uncommon but underappreciated fungal infection seen mostly in immunocompetent people, caused by the fungi Blastomyces. Blastomyces dermatitidis and Blastomyces gilchristii are the most common species responsible for most infections in the United States of America. Blastomycosis can cause a wide variety of diseases, the most common being pneumonia as the lungs are usually the main primary entrance source of infection after inhalation of Blastomyces spores. However, dissemination from the lungs to other organs such as skin, bones, genitourinary, and central nervous system can occur. We describe a 52-year-old female who was admitted to the hospital for unresolved pneumonia after failure to respond to multiple outpatient antibiotic treatments for a presumed diagnosis of community-acquired pneumonia, presenting with right-sided chest pain, fever, cough and weight loss for three weeks duration. Her initial Chest-X ray and CT chest revealed right middle lobe infiltrate, while her laboratory studies were significant for elevated white blood cells (WBC). The patient underwent diagnostic bronchoscopy which showed erythema of the right middle lobe bronchus with purulent secretion. Furthermore, cytology examination of specimens from bronchoalveolar lavage and bronchial brush from right middle lobe with (PAP) satin on thin prep and H&E stain on the cell block revealed an abundance of broad-base budding yeast consistent with Blastomyces organism, which was confirmed by molecular testing with DNA probe assay. Her serum and urine Blastomyces antigens were positive, above the limit of quantification. The patient was started on antifungal therapy with Amphotericin-B intravenously. In the process, her fever resolved and her respiratory symptoms slowly improved. She was discharged home with oral itraconazole which was to be continued for 6-12 months as an outpatient.

Spectrum-Resolved Electrochemiluminescence to Multiplex the Immunoassay and DNA Probe Assay.

The investigation on electrochemiluminescence (ECL) multiplexing bioassays mainly focuses on simultaneously detecting either proteins or nucleic acids. To overcome the limitation of a short waveband for spectrum-resolved ECL multiplexing bioassays, herein, a highly monochromatic (FWHM <40 nm) and bandgap-engineered ECL luminophore, that is, mercaptopropionic acid-capped and Zn2+-mediated aggregation-induced emission (AIE) assembly of Au nanocrystals (NCs) (Zn2+-AIE-AuNCs), of strong emission and the maximum emission wavelength at 485 nm is developed. The highly monochromatic and bandgap-engineered ECL (485 nm) of Zn2+-AIE-AuNCs can multiplex with the single-waveband and surface-defect-involved ECL (775 nm) of dual-stabilizer-capped CuInS2@ZnS NCs (CIS@ZnS-NCs), enabling a spectrum-resolved ECL multiplexing strategy with different NCs luminophores of a similar particle size as tags. This ECL multiplexing strategy can be utilized to simultaneously detect antigen and DNA probe together without any additional signal amplification procedure and obvious spectroscopic cross-talk, in which the highly monochromatic ECL from Zn2+-AIE-AuNCs is utilized to dynamically determine human carcinoembryonic antigen from 1 pg/mL to 50 ng/mL with a limit of detection (LOD) of 0.3 pg/mL, while the single-waveband ECL from CIS@ZnS-NCs is employed to linearly detect wild-type p53 from 1 pM to 50 nM with a LOD of 0.5 pM. The ECL immunoassay of the proposed strategy is free from the interference of the synchronously conducted DNA probe assay and vice versa, which would open an avenue to couple the immunoassay and DNA probe assay together for clinical colon and breast cancer identification.

Immunohistochemical detection of V600E BRAF mutation is a useful primary screening tool for malignant melanoma

ABSTRACTBackground: We compared the use of an immunohistochemical (IHC) method using a monoclonal antibody to BRAF V600E (which detects the main BRAF mutation) with existing DNA probe screening in tissue samples from 71 patients with malignant melanoma.Materials and methods: Paraffin blocks were cut to provide consecutive slides for haematoxylin and eosin staining, and for known positive micro-array DNA control material. IHC was performed by the Optiview detection system. All slides were scored independently by the clinical lead and the laboratory lead using a positive/negative system.Results: The DNA method found 26 samples to be positive, the IHC found 21 to be positive, giving a sensitivity value for IHC of 80.8%. However, all of the 45 samples found to be negative by DNA were also negative by IHC, giving a specificity of 100%. There were 66 instances of full agreement, giving a concordance of 93%. Together, these data give a kappa statistic of 0.843, indicating very good agreement.Conclusion: The data reveal a very close link between the two methods, supporting the use of the V600E as a primary screen for BRAF mutations in malignant melanoma. Samples found to be negative by this method may be retested by the DNA probe method. IHC detection conserves patient DNA from tumour blocks as only one section is required to perform the assay. The V600E antibody method is considerably cheaper and faster than the DNA probe assay, with a turn-around time of 24-48 hours, enabling more rapid clinical management.

Endonuclease IV based competitive DNA probe assay for differentiation of low-abundance point mutations by discriminating stable single-base mismatches.

We disclosed the unique discrimination property of Endo IV toward stable single-base mismatches located at the second nucleotide 3' to the AP site. Coupled with thermodynamic differentiation and competitive blocker strands, a highly sensitive and specific detection system was established with discrimination factors of 510-1079 for G:X mismatches and LODs of 0.003-0.005% for KRAS G12A, KRAS G12V and KRAS G12S mutations.

Printable Hecl Electrode Materials for Disposable Bioaffinity Assay Cartridges

We have demonstrated earlier that hot electrons can be injected into aqueous electrolyte solution from thin insulating film coated cathodes [1, 2]. Insulating film have typically been fabricated from an insulator material with a band gap of ca. 5 eV or preferably higher, with the Fermi level of the insulator somewhere in the mid band gap region. The hot electrons are emitted to aqueous electrolyte solution by direct field-assisted tunneling at these devices [2, 3]. After injection into the conduction band of water the hot electrons are thermalized and solvated to form hydrated electrons which finally act as reducing mediators in the Hot Electron-induced Electrochemiluminescence (HECL) systems. HECL is utilized mainly in immunoanalysis and in DNA-probe assays in which electrochemiluminescent labels are utilized [ 2 - 5 ]. Best labels are certain aromatic lanthanide chelates which allow sensitive time-resolved HECL detection of the labels [3, 6]. Silicon electrodes or silicon chips on which the working electrode/electrodes and counter electrode have been integrated have been shown to be excellent functional components of disposable assay cartridges [5,7]. Recently, we have been able to develop printable HECL electrode materials that show almost equal performance and are based on conductor particles embedded in polystyrene or in some other organic insulating materials.

Electrogenerated chemiluminescence induced by sequential hot electron and hole injection into aqueous electrolyte solution

Hole injection into aqueous electrolyte solution is proposed to occur when oxide-coated aluminum electrode is anodically pulse-polarized by a voltage pulse train containing sufficiently high-voltage anodic pulses. The effects of anodic pulses are studied by using an aromatic Tb(III) chelate as a probe known to produce intensive hot electron-induced electrochemiluminescence (HECL) with plain cathodic pulses and preoxidized electrodes. The presently studied system allows injection of hot electrons and holes successively into aqueous electrolyte solutions and can be utilized in detecting electrochemiluminescent labels in fully aqueous solutions, and actually, the system is suggested to be quite close to a pulse radiolysis system providing hydrated electrons and hydroxyl radicals as the primary radicals in aqueous solution without the problems and hazards of ionizing radiation. The analytical power of the present excitation waveforms are that they allow detection of electrochemiluminescent labels at very low detection limits in bioaffinity assays such as in immunoassays or DNA probe assays. The two important properties of the present waveforms are: (i) they provide in situ oxidation of the electrode surface resulting in the desired oxide film thickness and (ii) they can provide one-electron oxidants for the system by hole injection either via F- and F+-center band of the oxide or by direct hole injection to valence band of water at highly anodic pulse amplitudes.

(Invited) New Electrochemiluminescent Materials for Bioanalysis

Electrochemiluminescence (ECL), or electrogenerated chemiluminescence, is light-emitting phenomenon from electrochemical reactions. It has broad applications, such as immunoassays, DNA probe assays, aptasensors, biological study, drug screening, medicine determination, and light-emitting devices. Various ECL systems, such as ruthenium complex, luminol, semiconductor nanocrystals, graphene oxide, and nanoclusters, have been reported. Generally, ECL reactions involve in luminophores, coreactants, and electrode. Electrocatalysts and/or additives are also frequently used. The performance of ECL depends significantly on these materials used [1-2]. We have recently developed various materials to improve ECL performance, such as new coreactants, new luminophores, noble metal nanocrystals, silica nanomaterials, new electrodes. The possible mechanism for enhanced properties are discussed. The analytical applications of some materials were demonstrated. The use of these materials extends applications, improve ECL sensitivity and simplify detection. [3-7]. Acknowledgements This project was supported by the National Natural Science Foundation of China (No. 21475123 and 21175126).

Novel Helicobacter pylori sequencing test identifies high rate of clarithromycin resistance.

Eradication therapy selection for Helicobacter pylori gastritis requires knowledge of the local resistance rate to clarithromycin. There is minimal population-based or regional data in the United States on pediatric clarithromycin resistance. Although commercial methods such as fluorescence in situ hybridization and DNA probe assays are available in Europe for the evaluation of H pylori 23S rRNA mutations associated with resistance, clinical testing for 23S rRNA in the United States is not widely available. This study examined a single pediatric institution's clarithromycin resistance rate by a DNA polymerase chain reaction/sequencing assay applied to archived gastric biopsy specimens. From the period 2010 to 2012, 38 H pylori-infected gastric biopsies were examined from archived formalin-fixed paraffin-embedded (FFPE) material. The 23S rRNA gene of H pylori was polymerase chain reaction amplified and sequenced for the identification of point mutations that are associated with clarithromycin therapeutic resistance. By 23S rRNA gene sequencing, 50% (n=19) of the specimens contained H pylori with mutations significant for clarithromycin resistance. This study is consistent with other pediatric reports suggesting significant H pylori clarithromycin resistance in the United States. Furthermore, the method used in this study can be used by hospital-based clinical laboratories to assess local clarithromycin resistance from archived biopsy material.

Gastrointestinal Histoplasmosis in a Hepatitis C-Infected Individual

Gastrointestinal histoplasmosis is a rare manifestation of this fungal infection, typically identified in immunocompromised patients, such as those with HIV/AIDS. Here, we report a case of disseminated histoplasmosis with gastrointestinal involvement in a Hepatitis C-infected patient. The fungal agent was confirmed to be Histoplasma capsulatum by a DNA probe assay performed on a bone marrow sample. We propose that this fungal disease should be kept on the differential of patients infected with the Hepatitis C virus, as it has been reported to have numerous damaging effects on the adaptive immune system.

Luminometer development in the last four decades: recollections of two entrepreneurs

This month's featured review article; “Luminometer development in the last four decades: recollections of two entrepreneurs” is authored by Fritz Berthold and Veikko Tarkkanen. The review describes how coordinated R∓D activities in biochemistry and optical instrument development resulted in substantial applications of ATP bioluminescence in the food industry and beyond, including the design of more than 20 luminometers for immunoassays, DNA probe assays, and reporter gene assays. The cover of this issue of Luminescence features images of luminometers from the past four decades.

Delay to formalin fixation ‘cold ischemia time': effect on ERBB2 detection by in-situ hybridization and immunohistochemistry

The American Society of Clinical Oncology/College of American Pathologists ERBB2 testing guidelines address several pre-analytical variables known to affect ERBB2 testing accuracy. According to 2010 updated guidelines, the pre-analytical variable of time to tissue fixation (cold ischemia time) should be kept to <1 h, however, little has been published about cold ischemia time and its significance in ERBB2 testing. To that end, this study evaluated ERBB2 status using two different FDA-approved in-situ hybridization methods and an FDA-approved immunohistochemistry (IHC) assay in the largest cohort to date (n=84) of invasive breast carcinomas with tracked cold ischemia time. Cold ischemia time was stratified into four groups (<1 h (n=45), 1–2 h (n=27), 2–3 h (n=6), and >3 h (n=6)) and ERBB2 status was evaluated in each group by IHC (4B5) and by in-situ hybridization methodologies (PathVysion® fluorescence in situ hybridization and the INFORM HER2® dual in situ DNA probe assay). Both in-situ hybridization methods were evaluated using three ERBB2 scoring criteria (dual-probe guidelines, single-probe guidelines, and the FDA package insert scoring instructions). Fluorescence in-situ hybridization (FISH) and INFORM HER2® demonstrated 100% concordance in the detection of ERBB2 amplification by all three scoring guidelines at all cold ischemia time points. Agreement between in-situ hybridization methodologies and IHC was superior using single-probe guidelines compared with dual probe or FDA scoring instructions. In addition, Inform HER2®in-situ hybridization signals were significantly more intense than FISH at all cold ischemia time points, however, no significant loss of either chromosome 17 or ERBB2 signal was detected by FISH or Inform HER2®in-situ hybridization in cold ischemia times up to 3 h. On the basis of our findings, cold ischemia time up to 3 h has no deleterious effect on the detection of ERBB2 via in-situ hybridization or IHC.

L3.1 The Challenges of Drug-Diagnostic (RX-DX) Co-Development: A Biopharmaceutical Perspective

ABSTRACT There has been a major shift from conventional tumor histology alone to a biologically defined stratified approach that has led to the development of new cancer therapies and improved outcomes for cancer patients. The increasing technological power to interrogate the genome and other biologic processes, combined with sophisticated informatics, will continue to provide new biomarkers for targeted cancer therapies. While these factors are accelerating targeted cancer therapy, there are a large number of practical issues that need to be considered in the context of biopharmaceutical development and the marketing of new therapies that require a companion Dx. A Rx-Dx companion “product “should provide for appropriate therapy decision-making in the context of medical need. The strength of evidence for this typically comes from prospective clinical validation studies. Assay performance that meets technical analytical and classification accuracy criteria (including cut-off determination to define biomarker positive vs. negative) is critical for the prediction strength of the biomarker-endpoint relationship. Value creation is another important consideration for BioPharmaceutical and Diagnostic companies. This can be measured in terms of Rx or Dx development productivity, return on investment, and costs/benefits to the health care system. A Dx product intended to identify patients for a specific Rx therapy is a complex system of procedures, reagents, instruments, software, and algorithms, all of which may comprise the regulated product. A typical Dx development process includes discovery and feasibility research, product development, product verification and validation and regulatory approvals and commercialization. There are different regulatory expectations for Dx products across the world. The BioPharmaceutical company needs to integrate the Dx development and regulatory requirements with that of the Rx development and regulatory requirements. The recent approvals of vemurafenib with its companion cobas V600 mutation test and of crizotinib with its companion ALK FISH DNA probe assay are examples of how this can be achieved. Recognizing and managing the practical considerations, challenges and time required are critical to enabling successful Rx-Dx companion development to achieve the mission of delivering targeted health care to cancer patients.

A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni

A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10CFU for isolate cultures and 50CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4×102CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars.

Renal Failure Associated with APECED and Terminal 4q Deletion: Evidence of Autoimmune Nephropathy

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator gene (AIRE). Terminal 4q deletion is also a rare cytogenetic abnormality that causes a variable syndrome of dysmorphic features, mental retardation, growth retardation, and heart and limb defects. We report a 12-year-old Saudi boy with mucocutaneous candidiasis, hypoparathyroidism, and adrenocortical failure consistent with APECED. In addition, he has dysmorphic facial features, growth retardation, and severe global developmental delay. Patient had late development of chronic renal failure. The blastogenesis revealed depressed lymphocytes' response to Candida albicans at 38% when compared to control. Chromosome analysis of the patient revealed 46,XY,del(4)(q33). FISH using a 4p/4q subtelomere DNA probe assay confirmed the deletion of qter subtelomere on chromosome 4. Parental chromosomes were normal. The deleted array was further defined using array CGH. AIRE full gene sequencing revealed a homozygous mutation namely 845_846insC. Renal biopsy revealed chronic interstitial nephritis with advanced fibrosis. In addition, there was mesangial deposition of C3, C1q, and IgM. This is, to the best of our knowledge, the first paper showing evidence of autoimmune nephropathy by renal immunofluorescence in a patient with APECED and terminal 4q deletion.

Progress in Ru(bpy)32+ Electrogenerated Chemiluminescence

Abstract Among various electrogenerated chemiluminescence (ECL) systems, such as 9,10-diphenylanthracene, lucigenin, tris(2,2′-bipyridyl)ruthenium (Ru(bpy)32+), peroxyoxalate, luminol, graphene and nanocrystals, Ru(bpy)32+ ECL is one of the most widely applied ECL systems in recent years due to its broad applications in immunoassays, DNA probe assays, coreactants analysis and aptasensors. In this review, progress in Ru(bpy)32+ ECL has been summarized on the whole, and the future research trend has been proposed.

Impact of a Chemistry-Based DNA Extraction Method on Performance of a Commercial Amplification Assay for Detection of Mycobacterium tuberculosis Complex

Impact of a Chemistry-Based DNA Extraction Method on Performance of a Commercial Amplification Assay for Detection of <i>Mycobacterium tuberculosis</i> Complex

Intrathoracic Lymphadenitis Caused by Mycobacterium avium and Mycobacterium tuberculosis in an Immunocompetent Child

By means of a DNA probe assay (INNO-LiPA) we identified 2 different mycobacterial strains (Mycobacterium avium and Mycobacterium tuberculosis complex) from a mediastinal lymph node biopsy obtained from an apparently immunocompetent 7.5-year-old girl, whereas culture grew only M. avium. Clinicians should be aware of the possible occurrence of mixed infection involving both nontuberculous mycobacteria and M. tuberculosis.

Study of hepatocellular carcinoma type C by quantitation of serum HCV-RNA using branched DNA probe assay

Serum HCV-RNA levels were determined by newly developed branched DNA probe (bDNA) assay in 87 patients with hepatocellular carcinoma (HCC), compared with 73 patients with chronic hepatitis active and 30 patients with liver cirrhosis. Patients with decompensated liver cirrhosis (LC-d) had a significant lower viremia (mean 3.2Meg.eq./ml, bDNA positive rate; 40%) than chronic hepatitis active (18.0; 64%) and compensated LC (LC-c, 17.9; 80%) (p < 0.05). Those data indicates that HCV replication may decrease as progression of LC. In contrast, there was no difference between the levels of HCC with LC-c (8.2; 85%) and LC-d (6.1; 89%), and their positive rate of bDNA assay were significantly higher than LC-d without HCC (p < 0.01). Therefore, patients undergoing LC in whom serum HCV-RNA sustained high level may correspond to the high risk group of HCC. In HCC of heavy drinker, serum HCV-RNA levels (11.3; 91%) were significantly higher than the levels (4.6; 79%) of HCC without heavy drink (p < 0.05). The result indicates that heavy drink may induced an increase in HCV replication.

Determination of Total Calcium in Plasma by Flow Injection Analysis with Tris(2,2′‐bipyridyl)ruthenium(II) Electrochemiluminescent Detection

AbstractWe described here a new method for the determination of total calcium in plasma. The method is based on the precipitation of calcium with excess oxalate and the measurement of residual oxalate by flow injection analysis with Ru(bpy)32+ electrochemiluminescent detection. It has the advantages of extremely stable reagent, user‐friendly instrument, high selectivity, good analytical recovery, wide dynamic range, and nice correlation with atomic absorption spectroscopy. The calibration plot for calcium is linear over a concentration range from 0.5 mmol L−1 to 4.8 mmol L−1, which is wider than those obtained by most other methods. The analytical recoveries for plasma calcium are 98.4–101.2% with coefficients of variation (CVs) of 1.96–2.52%. The within‐day CVs range from 0.76% to 0.95%, and between‐day CVs were from 1.12% to 1.46%. The time for each injection is one minute. Because the proposed method can be readily carried out on increasingly popular instruments for Ru(bpy)32+ ECL immunoassays and DNA probe assays, Ru(bpy)32+ ECL method is suitable for routine clinical analysis of calcium.