Sensitive DNA Detection Research Articles

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

Multi intercalators FRET enhanced detection of minute amounts of DNA.

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.

S9.6 Antibody-Enzyme Conjugates for the Detection of DNA-RNA Hybrids.

Diagnosis of infectious agents is increasingly done by the detection of unique nucleic acid sequences, typically using methods such as PCR that specifically amplify these sequences. A largely neglected alternative approach is to use antibodies that recognize nucleic acids. The unique monoclonal antibody S9.6 recognizes DNA-RNA hybrids in a largely sequence-independent manner. S9.6 has been used in several cases for the analysis of nucleic acids. Extending our recent determination of the structure of S9.6 Fab bound to a DNA-RNA hybrid, we have developed reagents and methods for the sensitive detection of specific DNA and RNA sequences. To facilitate the use in diagnostics, we conjugated the S9.6 Fab to the highly active and well-characterized reporter enzyme human-secreted embryonic alkaline phosphatase (SEAP). Two approaches were utilized for conjugation. The first used sortase A (SrtA), which generates a covalent peptide bond between short amino acid sequences added to recombinantly produced S9.6 Fab and SEAP. The second approach was to genetically fuse the S9.6 Fab and SEAP so that the two are produced as a single molecule. Using these two antibody-SEAP proteins, we developed a simplified ELISA format for the identification of synthetic DNA-RNA hybrids, which can be optimized for detecting nucleic acids of pathogens, as well as for other applications. We successfully used this immunosorbent assay, HC-S, to identify DNA-RNA hybrids in solution with high specificity and sensitivity.

Comparison of different noble metal-based screen-printed sensors for detection of PIK3CA point-mutations as biomarker for circulating tumor DNA

Screen-printed electrochemical sensors are of particular interest for point-of-care diagnostic applications due to their high sensitivity, short analysis times, and cost-efficient large-scale fabrication. Especially noble metal-based sensors offer the possibility of easy surface modification via self-assembly of thiol-labelled compounds. We examined silver, gold, and palladium as electrode materials regarding their suitability for highly specific and sensitive DNA detection with a chronoamperometric enzyme-amplified electrochemical assay. Modification with either gold nanoparticles or silver-coated microbeads was tested as a possibility to improve the performance of the electrochemical sensors. The sensors were characterized regarding surface topography, sensitivity towards the redox-active substrate TMB, efficiency of capture probe immobilization, as well as mutation specificity. The highest sensitivity for oxidized TMB was observed for gold sensors that yielded a limit of detection of 2.1 µM, while no improvement in sensitivity was observed for sensors modified with gold nanoparticles or silver-coated microbeads. DNA probe immobilization and multiplexed target DNA detection was successful with all tested electrode materials. However, modification with silver-coated microbeads led to both reduced probe immobilization efficiency and hybridization specificity. Gold nanoparticle modification of both silver and of gold sensors induced a highly porous sensor surface after sintering, which increases the surface area of the electrodes. The gold nanoparticle modified sensors showed particularly appreciable results in terms of improved hybridization specificity, as demonstrated by successful multiplexed detection of three PIK3CA point-mutations (H1047R, E545K, and E542K). Average discrimination factors D of 2.7 and 2.1 were obtained for gold and silver sensors modified with one layer of gold nanoparticles, respectively.

Modified gold nanoparticle colorimetric probe-based biosensor for direct and rapid detection of Mycobacterium tuberculosis in sputum specimens

The incidence of Mycobacterium tuberculosis (MTB) is increasing due to lack of appropriate diagnostic and therapeutic methods. Therefore, early and accurate detection of this bacteria plays a significant role in controlling tuberculosis. This study aimed to design, develop, and implement a direct and rapid detection method of MTB using modified gold nanoparticle (AuNP) colorimetric probe-based biosensor in sputum specimens. Spherical AuNPs were synthesized by the citrate reduction method and were functionalized using thiol-modified oligonucleotides (AuNP-biosensor). AuNP-biosensor and IS6110 PCR were compared to the gold standard in terms of analytical and clinical sensitivity and specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and accuracy in 52 clinical specimens. Gold standard was defined as a positive result in concentrated sputum smear microscopy (SSM), culture, or Xpert MTB/RIF.The AuNP-biosensor had 100% sensitivity and specificity for detection of total sputum DNA in less than 15min with ready-to-use AuNP-biosensor. PPV, NPV, DOR and accuracy of this method were 100%, 100%, 2325 and 100%, respectively. Considering the promising results of the diagnostic value indices of the AuNP-biosensor, the designed method is an affordable, rapid, reliable, and cost-beneficial way for direct detection of MTB in sputum specimens.

A CRISPR/Cas12a-Mediated Sensitive DNA Detection System for Gene-Edited Rice.

In recent years, genome editing technology represented byclustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing. We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products. In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods. The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especiallyfor low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products. We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice. The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.

Evaluation of the efficacy of tenofovir disoproxil fumarate in the treatment of chronic hepatitis B combined with nonalcoholic fatty liver disease by high-sensitivity detection of HBV DNA levels

Objective: To observe the efficacy of tenofovir disoproxil fumarate (TDF) antiviral therapy in patients with chronic hepatitis B (CHB) combined with nonalcoholic fatty liver disease (NAFLD), so as to provide evidence-based evidence in these special populations. Methods: Data from 91 CHB cases who received TDF 300 mg/d antiviral therapy for 96 weeks were analyzed retrospectively. Among them, 43 cases with NAFLD were included in the study group, and 48 cases without NAFLD were included in the control group. The virological and biochemical responses of the two groups of patients at 12, 24, 48, and 96 weeks were compared. Among them, 69 patients underwent highly sensitive detection of HBV DNA. The t-test and χ (2) test were performed on the data. Results: ALT normalization rate was lower in the study group (42%, 51%) at 12 and 24 weeks of treatment than that in the control group (69%, 79%), and the difference was statistically significant (P < 0.05). However, there was no statistically significant difference between the two groups at 48 and 96 weeks. HBV DNA concentration below the lower limit of detection (200 IU/ml) was lower in the study group at 12 weeks of treatment than in the control group (35% vs. 56%), and the difference was statistically significant (P < 0.05). However, there was no statistically significant difference between the two groups at 24, 48, and 96 weeks. Furthermore, HBV DNA concentration below the lower limit was significantly lower in the study group than that in the control group at 12, 24, 48, and 96 weeks of treatment when the lower limit of HBV DNA detection was set at 20IU/ml, and the difference was statistically significant (P < 0.05). The HBeAg serological negative conversion rate was gradually higher in the study group at 48 and 96 weeks of treatment than in the control group, and the difference was not statistically significant. Conclusion: TDF antiviral treatment can affect the virological and biochemical responses of NAFLD in chronic hepatitis B.

Sensitive Detection of Cell-Free Tumour DNA Using Optimised Targeted Sequencing Can Predict Prognosis in Gastro-Oesophageal Cancer.

In this longitudinal study, cell-free tumour DNA (a liquid biopsy) from plasma was explored as a prognostic biomarker for gastro-oesophageal cancer. Both tumour-informed and tumour-agnostic approaches for plasma variant filtering were evaluated in 47 participants. This was possible through sequencing of DNA from tissue biopsies from all participants and cell-free DNA from plasma sampled before and after surgery (n = 42), as well as DNA from white blood cells (n = 21) using a custom gene panel with and without unique molecular identifiers (UMIs). A subset of the plasma samples (n = 12) was also assayed with targeted droplet digital PCR (ddPCR). In 17/31 (55%) diagnostic plasma samples, tissue-verified cancer-associated variants could be detected by the gene panel. In the tumour-agnostic approach, 26 participants (59%) had cancer-associated variants, and UMIs were necessary to filter the true variants from the technical artefacts. Additionally, clonal haematopoietic variants could be excluded using the matched white blood cells or follow-up plasma samples. ddPCR detected its targets in 10/12 (83%) and provided an ultra-sensitive method for follow-up. Detectable cancer-associated variants in plasma correlated to a shorter overall survival and shorter time to progression, with a significant correlation for the tumour-informed approaches. In summary, liquid biopsy gene panel sequencing using a tumour-agnostic approach can be applied to all patients regardless of the presence of a tissue biopsy, although this requires UMIs and the exclusion of clonal haematopoietic variants. However, if sequencing data from tumour biopsies are available, a tumour-informed approach improves the value of cell-free tumour DNA as a negative prognostic biomarker in gastro-oesophageal cancer patients.

Amplification-free detection of HBV DNA mediated by CRISPR-Cas12a using surface-enhanced Raman spectroscopy

Nucleic acid markers have been widely used in the detection of various virus-related diseases, including hepatitis B virus (HBV), which is spreading worldwide. The trans-activated CRISPR-Cas system has shown excellent sensitivity and specificity in nucleic acid detection. However, nucleic acid testing usually requires amplification of the target nucleic acid for more accurate and specific detection; furthermore, current nucleic acid assays are time-consuming, costly, and are limited by non-specific cross-reactivity. We developed an amplification-free viral DNA biosensor-based diagnostic method that uses a clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas)-based approach with surface enhanced Raman spectroscopy. This method can specifically identify the target site by changing the crRNA sequence. In addition, the incubation period and development of the disease can be determined by quantitative detection of viral DNA. This system could achieve rapid and highly sensitive detection of HBV DNA within 50 min and vast detection range from 0.1 pM to 1 nM. Therefore, a combined CRISPR/Cas12a-SERS-based assay would improve the sensitivity of detection in assays using multiple biomarkers. In conclusion, our CRISPR/Cas12a-based biosensor would enable rapid, simple, and sensitive detection of HBV nucleic acids.

Label-free electrochemical detection of cancer biomarkers DNA and anti-p53 at tin oxide quantum dot-gold-DNA nanoparticle modified electrode

Lung cancer is one of the deadliest types of cancer and accounts for 8.1% of all cancer related deaths. To prevent a growing death rate, it is crucial to identify lung cancer at an early stage by single polynucleotide morphism detection. In this paper, we present a novel label-free electrochemical biosensor based on composites of tin oxide quantum dots and gold nanoparticles (SnO2-QD-Au) for the sensitive and precise detection of lung cancer DNA. The SnO2-QD and SnO2-QD-Au nanoparticles were characterized using Scanning and Transmission Electron Microscopes (SEM and TEM), X-ray diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), UV–Vis spectroscopy (UV), Fourier transmission infrared spectroscopy (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Gold thiol covalent bonding was used for immobilising probe DNA on the surface of SnO2-QD-Au nanoparticles followed by target DNA hybridization and detected electrochemically in presence of 1 mM [Fe(CN6)]3−/4−as a redox couple probe. Under ideal circumstances, the sensor showed the lowest detection limit of 3.2 × 10−20 M with a linear range of 1 × 10−6 − 1 × 10−20 M. Additionally, the sensing method was applied to find a cancer biomarker, Anti-P53 antibody.

AuPt nanoalloy with dual functionalities for sensitive detection of HPV16 DNA.

Human papillomavirus type 16 (HPV16), one of the high-risk types, is responsible for 53% of cervical cancers. The development of an early diagnostic approach with high sensitivity, low-cost, point-of-care testing (POCT) for HPV16 is urgent. In our work, a novel dual-functional AuPt nanoalloy-based lateral flow nucleic acid biosensor (AuPt nanoalloy-based LFNAB) was established with excellent sensitivity for detecting HPV16 DNA for the first time. The AuPt nanoalloy particles were prepared by a one-step reduction method, which was simple, rapid, and green. The AuPt nanoalloy particles retained the performance of initial Au nanoparticles owing to the catalytic activity enabled by Pt. Such dual-functionalities offered two kinds of detection alternatives (i.e., normal mode and amplification mode, respectively). The former is produced just by the black color from the AuPt nanoalloy material itself, and the latter is more color sensitive from its superior catalytic activity. The optimized AuPt nanoalloy-based LFNAB exhibited satisfactory quantitative ability in detecting the target HPV16 DNA in the range of 5-200 pM with a LOD of 0.8 pM at the "amplification mode". The proposed dual-functional AuPt nanoalloy-based LFNAB displayed great potential and promising opportunity in POCT clinical diagnostics.

A ratiometric fluorescent biosensor for rapid detection of Burkholderia pseudomallei by dual CRISPR/Cas12a trans-cleavage assisted signal enhancement

Melioidosis caused by Burkholderia pseudomallei (Bp) infection has high mortality in tropical and subtropical regions. A rapid and sensitive method for its detection is urgently needed. Herein, a ratiometric fluorescent sensor with dual recognition sites of target was designed for sensitive detection of Bp DNA. The sensor was constructed with a TAMRA-labeled DNA as probe, which could adsorb on polymer dots (Pdots) at acidic pH to produce TAMRA fluorescence at the excitation wavelength of Pdots via fluorescence resonance energy transfer (FRET). In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a was activated with the dual recognition sites, which digested the probe DNA to weaken the FRET. With the fluorescence ratio of Pdots to TAMRA as the detection signal, a ratiometric fluorescent biosensing was thus achieved. This method with dual CRISPR/Cas12a trans-cleavage assisted signal enhancement showed a detectable range of target DNA from 1.0 pM to 10 nM, and could be successfully used to detect Bp DNA extract with a detection time of 40 min. The excellent performance such as high sensitivity, good specificity, acceptable accuracy and short analytical time along with convenient operation demonstrated its potential in clinic diagnosis of melioidosis.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus

Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10−6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

Rose bengal-mediated photoinduced atom transfer radical polymerization for high sensitivity detection of target DNA

Convenient and sensitive detection of biomolecules is of utmost importance in the field of early disease screening. In this study, a Rose Bengal-Mediated photoinduced atom transfer radical polymerization (photoATRP) method was used to achieve highly sensitive detection of target DNA (tDNA). The tDNA was specifically recognized using PNA with terminal modified sulfhydryl groups, and the initiator α-bromophenylacetic acid (BPAA) was attached to the electrode surface via a phosphate-Zr4+-carboxylate acid structure. Under the excitation of blue light, rose bengal (RB) acts as a photocatalyst, β-nicotinamide adenine dinucleotide (NADH) as an electron donor, and ferrocenylmethyl methacrylate (FMMA) as a monomer to activate the photoATRP reaction and generate a large number of electroactive polymer chains on the electrode surface. Under optimal conditions, the method can be used for the quantitative analysis of tDNA in the concentration range of 1–105 fM (R2 = 0.994) with a limit of detection (LOD) of 0.115 fM. This metal-free mediated photoATRP biosensor, with low cost and environmental friendliness, has great potential in the field of highly sensitive biomolecule detection.

Laser-Induced graphene electrodes for highly sensitive detection of DNA hybridization via consecutive cytosines (polyC)-DNA-based electrochemical biosensors

Numerous carbon-based biosensors issued mechanical exfoliation, epitaxial growth, reduced graphene oxide, and chemical vapor deposition have been investigated for highly sensitive and specific detection of DNA. As a promising route for designing electrochemical biosensor-based flexible substrates, the laser-induced graphene technique, which provides a cheap, technologically simple, and highly robust sensing platform, has been widely adopted. However, DNA-based biosensors' efficiency is strongly dependent on how DNA probes are tethered to the nanomaterials. In view of this, poly-cytosine (poly-C) DNA has shown outstanding adsorption to multiple inorganic nanomaterials, including gold (Au), zinc oxide (ZnO), tungsten disulfide (WS2), graphene oxide (GO), and graphene. In this work, a poly C(15)-tailed diblock DNA probe is used to anchor to carbonized working electrode issued laser-induced method. Meanwhile, the second block modified with ferrocene (Fc) derivatives is lifted at the surface for DNA sequence recognition. Following this strategy, the developed biosensor leads to a limit of detection (LOD) of 57 fM, which was superior or comparable to some previously reported methods. Moreover, the proposed electrochemical DNA biosensor exhibits high specificity in differentiating the complementary DNA from non-complementary DNA (ncDNA), and mismatched DNAs (MM-DNA) sequences. Finally, the easily constructed laser-induced graphene electrode biosensor showed an ability to detect DNA in human serum as a complex environment, making our approach a promising avenue for disease diagnosis.

BIFLUOROPHORES BASED ON NAPHTHALIC ACID DERIVATIVES AS BIOLOGICALLY ACTIVE SUBSTANCES

A large number of organic compounds, capable to luminesce under influence of various types of radiation, are known nowadays. These substances are called luminophores. Features of the chemical structure of these compounds determine their luminescent properties and behavior of a specific luminophore molecules in one or another environment. Possessing a number of spectral properties, luminophores are widely used in many spheres of human a ctivity. Their usage in biology and medicine plays an important role. Number of new methods of biological research, developed on the basis of luminescent dyes, is growing very quickly. This explains the need for synthesis of new luminescent dyes. Bright and water-soluble fluorescent dyes, for example, are important for sensitive detection of DNA via fluorescent probes. Currently, search for new potential antiviral agents is divided in two main strategies. One trend concerns synthesis of new derivatives of already existing antiviral drugs; chemical synthesis of such compounds is aimed at creating a new, more effective drug. Another trend in work on new antiviral drugs is the search for natural compounds, mainly of plant origin, in order to obtain potential chemotherapeutic agents. An interesting object of synthesis is formazans -- a class of organic substances with various biological activities.Biological and industrial application of formazans begins with their use as analytical reagents and dyes, and ends, primarily interesting for us, with their usage in pharmacology. They found wide practical usage as dyes, indicators of redox reactions, etc. In medicine, formazans are used due to their antioxidant, antiviral, antimicrobial, analgesic, anthelmintic and other properties. Many formazans are used in medicine as indicators of cancer cells, drugs against tuberculosis, influenza, and fungal diseases. Important advantage in of formazans synthesis is fairly easy and cheap methods of their production, available starting materials and speed of synthesis processes. In addition, an interesting direction in search for effective substances is the synthesis of formazans with luminescent properties, which significantly expand areas of their application. In our case, such organic luminophores are formazanonaphthalimides and formazanoacenaphthenquinones, which are close to them in structure.

Sensitive DNA Detection using a Branched DNA as a Sensor Coupled with Hybridization Chain Reaction

AbstractNucleic acids detection is becoming increasingly vital for disease's diagnosis. A sensitive, convenient and simple detection method provide the possibility of self‐diagnosis to prevent the disease's development. Herein, we designed a novel branched DNA nanostructure as probe, which could induce DNA hybridization chain reaction in the presence of target DNA. The strategy as amplifier enhanced the electrochemical signal of target DNA for sensitive nucleic acids detection. In this system, the branched DNA probe possessed more capturing points for Target DNA than previous single‐stranded DNA. Target DNA as trigger were used to induce hybridization chain reaction in three dimensions. The limit of detection of this strategy can reach to 1 pM with excellent specificity and selectivity. Meanwhile, the linear working range was from 10 pM to 100 μM. The biosensor had a great promising in the determination of nucleic acids for disease's diagnosis.

Detecting DNA damage in stored blood samples

Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.

Diagnosis and clinical significance of Human bocavirus 1 in children hospitalized for lower acute respiratory infection: molecular detection in respiratory secretions and serum.

Introduction. Human bocavirus 1 (HBoV1) infection occurs with viral genome presence in respiratory secretions (RS) and serum, and therefore both samples can be used for diagnosis.Gap statement. The diagnostic sensitivity of HBoV1 DNA detection in serum and the duration of DNAaemia in severe clinical cases have not been elucidated.Aim. To determine HBoV1 DNA in serum and RS of paediatric patients hospitalized for lower acute respiratory infection (LARI) and to analyse the clinical-epidemiological features of positive cases.Methodology. This was a prospective, transverse study. Physicians selected the clinical situations and obtained paired clinical samples (RS and serum) that were tested by PCR/qPCR for HBoV1. Positive cases were analysed considering time of specimen collection, co-detection, clinical manifestations and viral load; statistical significant level was set at α=0.05.Results. HBoV1 was detected in 98 of 402 cases included (24 %); 18/98 (18 %) patients had the virus detectable in serum and 91/98 (93 %) in RS (P<0.001). Positivity rates were not significantly different in patients with RS and serum collected within or beyond 24 h of admission. Single HBoV1 infection was identified in 39/98 patients (40 %), three patients had HBoV1 in both clinical samples (3/39, 8 %) and 32 (32/39, 82 %) only in RS, 22 of them (69 %) with both clinical samples within 24 h of admission. Cough (P=0.001) and rhinitis (P=0.003) were significantly frequent among them and most patients were diagnosed with bronchiolitis (22/39, 56 %) and pneumonia (9/39, 23 %), which was more frequent compared to cases with co-infection (P=0.04). No significant differences were identified among patients with high, medium or low viral load of HBoV1 regarding rate of positivity in both clinical samples, the time of collection of RS and serum, co-detection, first episode of LARI, clinical manifestations, comorbidity or requirement for assisted ventilation. Intensive care unit (ICU) patients had a significantly higher frequency of detection (P<0.001) and co-detection (P=0.001) compared to patients on standard care.Conclusions. HBoV1 is prevalent among infant patients hospitalized for LARI and including it in the standard testing can add to the aetiological diagnosis in these cases, especially for patients admitted to the ICU. HBoV1 detection in serum did not contribute significantly to the diagnosis as compared to detection in respiratory secretions.

Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies.

Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies.