Specific DNA Detection Research Articles

Abstract LB190: DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations

Abstract Genomic DNA anomalies such as copy number variations (gene duplication, amplification, deletion) and gene rearrangements are important biomarkers and drug targets in many cancer types. DNA in-situ hybridization (ISH) is the gold standard method to directly visualize these molecular alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissues at single-cell resolution within a histological section. However, currently available fluorescent ISH (FISH) assays provide limited morphological detail due to the use of fluorescent nuclear staining compared to chromogenic staining. Furthermore, FISH techniques rely on expensive fluorescence microscopes, risk loss of fluorescent signal over time and involve tedious imaging at high magnifications (100X). There is thus an unmet need for a sensitive and robust chromogenic DNA-ISH assay that can enable high-resolution detection of genomic DNA targets with the ease of bright-field microscopy. We present here DNAscope - a novel chromogenic DNA-ISH assay - for detecting and visualizing genomic DNA targets under a standard light microscope. DNAscope is based on the widely used RNAscope® double-Z probe design and signal amplification technology and provides unparalleled sensitivity and specificity with large signal dots readily visualized at 40X magnification and with full morphological context. Furthermore, DNAscope ensures specific DNA detection without interference from RNA due to the use of a novel RNA removal method. Using a duplex chromogenic detection assay in red and blue, we demonstrate highly specific and efficient detection of gene rearrangements (ALK, ROS1, RET and NTRK1), gene amplification (ERBB2, EGFR, MET) and deletion (TP53 and CDKN2A). The DNAscope assay has been carefully optimized for probe signal size and color contrast to enable easy interpretation of signal patterns under conventional light microscopy or digital pathology. Compared to conventional FISH assays, DNAscope probes are standard oligos that are designed in silico to be free of any repetitive sequences and can be rapidly synthesized for any DNA target. In conclusion, the DNAscope assay provides a powerful and convenient alternative to commonly used FISH assays in many cancer research applications. Citation Format: Li-Chong Wang, Farzaneh Tondnevis, Courtney Todorov, Jayson Gaspar, Aparna Sahajan, Vasudha Murlidhar, Bingqing Zhang, Xiao-Jun Ma. DNAscopeTM: A novel chromogenic in-situ hybridization technology for high-resolution detection of DNA copy number and structural variations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB190.

Detection of cell-free microbial DNA using a contaminant-controlled analysis framework

BackgroundThe human microbiome plays an important role in cancer. Accumulating evidence indicates that commensal microbiome-derived DNA may be represented in minute quantities in the cell-free DNA of human blood and could possibly be harnessed as a new cancer biomarker. However, there has been limited use of rigorous experimental controls to account for contamination, which invariably affects low-biomass microbiome studies.ResultsWe apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients.ConclusionsTogether, these observations indicate that plasma can harbour a low yet detectable level of cfmDNA. The results highlight the importance of accounting for contamination and provide an analytical decontamination framework to allow the accurate detection of cfmDNA for future biomarker studies in cancer and other diseases.

Double Hairpin DNAs Recognition Induced a Novel Cascade Amplification for Highly Specific and Ultrasensitive Electrochemiluminescence Detection of DNA.

Herein, a novel DNA cascade amplification, including double hairpin DNAs recognition-triggered single-target recycling (D-STR) and concatenated DNA structure-controlled rolling circle amplification (C-RCA), was developed as a signal amplifier to construct a highly specific and ultrasensitive electrochemiluminescence (ECL) biosensor for human immunodeficiency virus (HIV) DNA fragments detection, which not only revealed tremendous potential in avoiding false positive signals but also obviously promoted the amplification efficiency simultaneously compared to conventional single recognition of the target. Once the target DNA triggered the rolling circle amplification (RCA), the obtained RCA products could be anchored on the Pt-modified glassy carbon electrode (GCE) via the Pt-N bond, capturing massive ruthenium (Ru)-labeled ssDNA as the ECL signal tag to generate remarkable ECL emission. As a result, the proposed biosensor showed highly specific and ultrasensitive detection of the target with the detection limit down to 27.0 aM, which gives great impetus to the development of a novel specific biosensor for practical bioanalysis and diagnostic technologies.

Construction of a CRISPR-Biolayer Interferometry Platform for Real-Time, Sensitive, and Specific DNA Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA-recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real-time, sensitive, and rapid digital system called CRISPR-BLI was established for the detection of double-stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR-BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR-BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.

High-fidelity detection of DNA combining the CRISPR/Cas9 system and hairpin probe

Methods that enable specific and sensitive detection of DNA are greatly required for high-fidelity sequence measurement and single-nucleotide variations (SNVs) genotyping. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems have provided revolutionary tools for detecting nucleic acids. However, most of the current CRISPR/Cas-based DNA biosensing platforms suffer from inherent off-target effects of Cas proteins and require pre-amplification processes, which compromise the analytical fidelity. In this work, a CRISPR/Cas9-triggered hairpin probe-mediated biosensing method (namely CHP) was used to directly read the original DNA sequences, while effectively neutralizing the off-target effect and achieving high sensitivity. This technique can quantify DNA targets with a limit of detection (LOD) at the attomole level and identify SNVs with allelic fractions as low as 0.01%~0.1%. Moreover, we show that the CHP system is applicable in detecting mutations in serum samples without DNA isolation steps. Collectively, the CHP system is a sensitive and high-fidelity platform, which promises a great potential for providing robust tool for DNA sequence analysis and SNVs genotyping.

Periodically programmed building and collapse of DNA networks enables an ultrahigh signal amplification effect for ultrasensitive nucleic acids analysis

Analysisof molecular species is needed for applications in diagnosis of infections and genetic diseases. Herein, we demonstrate a target DNA-responsive ultrahigh fluorescence signal-on DNA amplification system via periodically programmed building and collapse of DNA networks. In this system, a pair of oligonucleotides of padlock probe (PP) and palindromic hairpin probe (PHP) are utilized. The presence of target DNA firstly hybridizes with PP, allowing the occurrence of rolling circle amplification (RCA) to produce RCA products with tandem repeats in abundance to bind and unfold numbers of PHPs. The conformational change of PHPs enables the building of DNA networks via the intermolecular palindrome pairing, but then makes the DNA networks collapsed via the palindrome-induced strand displacement polymerization. The displaced RCA products are dynamically reused to undergo periodically programmed multiple rounds of DNA network building and collapse. Depend on the bidirectional DNA assembly and disassembly, a strikingly amplified fluorescence can be collected to ultrasensitive and specific detection of target DNA. The practicability has been demonstrated by evaluating target-spiked human serum, saliva, and urine samples with acceptable recoveries and reproducibility. Therefore, this newly explored method opens a promising avenue for the detection of nucleic acids with low abundance in biochemical analysis and diseases diagnosis.

Utility of Fine Needle Aspiration Cytology (FNAC) in the diagnosis of tuberculous lymphadenitis compared to GeneXpert in a tertiary health care center in Northern Odisha, India

BackgroundDiagnosis of extrapulmonary tuberculosis including tuberculous lymphadenitis (TBLN) is challenging because of its atypical clinical presentation, paucibacillary nature of mycobacteria at the infected sites, variation in sensitivity of a test to specimens collected by different methods and from different infected tissues. MethodsIn the present study, suspected individuals for lymph node tuberculosis irrespective of age were enrolled prospectively and specimens were collected aseptically by fine needle aspiration (FNA). After the implementation of exclusion criteria, FNA specimens from a total of 278 cases of suspected TBLN were evaluated for cytomorphology (FNAC), presence of acid-fast bacillus (AFB) in smear microscopy and specific detection of mycobacterial DNA in cartridge-based nucleic acid amplification test (CBNAAT). ResultsThe results showed high prevalence of Type II (59.71%), followed by Type I (34.53%) and Type III (5.75%) pattern in FNAC. Non-type II patterns were significantly high in regions outside of the head and neck region (P = 0.031; OR = 2.125) and had an increasing trend of their occurrences with progression of age. The most affected age group was between 16 and 30 years with female preponderance documented in individuals below 45 years, whereas male preponderance was observed in higher age group patients, majority of whom had infected lymph nodes outside of HAN region (P = 0.063, OR = 1.998). The results also showed high sensitivity of CBNAAT (83.04%) method followed by FNAC (72.17%) with AFB smear exhibiting the disappointing results (sensitivity of 10.86%) compared to the CRS. High percentage of positivity was observed in Type III (AFB:25% vs CBNAAT: 100%) followed by Type II (AFB:10.2 vs CBNAAT: 76.5), while low detection was observed from samples with Type I (AFB:4.2 vs CBNAAT: 50). Interestingly, CBNAAT detection of TB was shown to be unaffected by gender, age and site of infection. ConclusionThe study suggests a possible contributary role of age and gender for cytomorphological pattern distribution of TBLN at various body parts. Although FNAC detected TB in 77.1% of cases which were identified positive by CBNAAT and/or AFB, it is being solely based on cytomorphology cannot be used alone as a reliable diagnostic method for TBLN detection. Further, the negative results in CBNAAT for FNAC positive cases may not necessarily be non-TB cases and must be evaluated by other diagnostic modalities. We recommend for both cytomorphological investigation and CBNNAT for the fine needle aspirates from suspected TBLN and subsequent treatment to reduce the disease burden.

Highly sensitive label-free fluorescence determination of lymphotropic virus DNA based on exonuclease assisted target recycling amplification and in-situ generation of fluorescent copper nanoclusters

Specific and sensitive detection of target DNA plays a significant role in clinical diagnosis and biomedical research. Herein, a sensitive ratio fluorescence biosensor was constructed based on in-situ generation of fluorescent copper nanoclusters (CuNCs) on the DNA modified graphene quantum dots (GQDs) for the detection of HTLV-I DNA. Firstly, large amount of AT rich single DNA (o-DNA) can be released from the designed hairpin DNA in the presence of trace target DNA via exonuclease III assisted target recycling amplification strategy. Then, the released o-DNA hybridized with the complementary DNA1 which was modified on the surface of GQDs, and the formed hybridized DNA with blunt 3′-hydroxylated terminus that couldn’t be digested by exonuclease I could act as the template for the generation of fluorescent CuNCs. While in the absence of target DNA, the o-DNA couldn’t be released, thus the DNA1 with 3′overhang ends could be degraded by exonuclease I, which resulted in the in-situ generation of fluorescent CuNCs on the surface of GQDs was blocked owing to the lack of DNA template. The GQD fluorescence in the GQD-CuNC nanohybrid was almost uninfluenced during the whole process. Therefore, with the GQD serving as the reference and CuNC acting as the reporter signal, there was an excellent linear relationship between the change of fluorescence intensity ratio (F595/F445) and the concentration of HTLV-I DNA in the range of 20 pM-12 nM with a detection limit of 10 pM. Furthermore, the biosensor exhibited satisfactory results for monitoring the presence of HTLV-I DNA in human serum.

DNA Footprints: Using Parasites to Detect Elusive Animals, Proof of Principle in Hedgehogs.

Simple SummaryNocturnal and elusive animals are notoriously difficult to count—hedgehogs being a prime example. Therefore, any reliable way to demonstrate the presence of a particular animal, within a given area, would be a valuable addition to many ecologists’ tool kits. The proposed method is based upon the idea that you can find a parasite, specific to a vertebrate animal of interest that has a life stage within an invertebrate host. Molecular detection of these parasites is then carried out in the more abundant and easily collected invertebrate intermediate host. The key to this proposed method is the specificity of the parasite to the vertebrate animal and its detection in the invertebrate intermediate hosts. Crenosoma striatum is specific to hedgehogs and was chosen as the parasite to develop the molecular survey tool for hedgehogs, an elusive nocturnal species of considerable interest at present. Results revealed the presence of the nematode only at a site known to be inhabited by hedgehogs confirming the potential of this method to improve the accuracy of recording hedgehog populations.The Western European Hedgehog (Erinaceous europaeus) is a nocturnal animal that is in decline in much of Europe, but the monitoring of this species is subjective, prone to error, and an inadequate basis for estimating population trends. Here, we report the use of Crenosoma striatum, a parasitic nematode specific to hedgehogs as definitive hosts, to detect hedgehog presence in the natural environment. This is achieved through collecting and sampling the parasites within their intermediate hosts, gastropoda, a group much simpler to locate and sample in both urban and rural habitats. C. striatum and Crenosoma vulpis were collected post-mortem from the lungs of hedgehogs and foxes, respectively. Slugs were collected in two sessions, during spring and autumn, from Skomer Island (n = 21), which is known to be free of hedgehogs (and foxes); and Pennard, Swansea (n = 42), known to have a healthy hedgehog population. The second internal transcribed spacer of parasite ribosomal DNA was used to develop a highly specific, novel, PCR based multiplex assay. Crenosoma striatum was found only at the site known to be inhabited by hedgehogs, at an average prevalence in gastropods of 10% in spring and autumn. The molecular test was highly specific: One mollusc was positive for both C. striatum and C. vulpis, and differentiation between the two nematode species was clear. This study demonstrates proof of principle for using detection of specific parasite DNA in easily sampled intermediate hosts to confirm the presence of an elusive nocturnal definitive host species. The approach has great potential as an adaptable, objective tool to supplement and support existing ecological survey methods.

Circulating Tumor DNA in KRAS positive colorectal cancer patients as a prognostic factor - a systematic review and meta-analysis.

Liquid biopsy is a novel tool in oncology. It provides minimally invasive detection of tumor specific DNA. This review summarizes data on presence of circulating tumor DNA in serum or plasma of CRC patients as a potential negative prognostic factor. The systematic review was performed according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). The search was performed using PubMed, Web of Science and Scopus. In total 18 articles with a total of 1779 patients met the inclusion criteria. Six out of 8 studies found that presence of ctDNA in plasma/serum was associated with inferior overall survival. All 6 studies found that high concentrations of ctDNA in plasma/serum was associated with inferior overall survival. Presence or high concentrations of KRAS mutation in plasma or serum were associated with inferior prognosis. Establishing cut-off concentrations is warranted for further clinical implementation of liquid biopsy.

Rapid and specific DNA detection by magnetic field-enhanced agglutination assay

The detection of DNA molecules by agglutination assays has suffered from a lack of specificity. The specificity can be improved by introducing a hybridization step with a specific probe. We developed a setting that captured biotinylated DNA targets between magnetic nanoparticles (MNPs) grafted with tetrathiolated probes and anti-biotin antibodies. The agglutination assay was enhanced using a series of magnetization cycles. This setting allowed to successfully detect a synthetic single stranded DNA with a sensitivity as low as 9 pM. We next adapted this setting to the detection of PCR products. We first developed an asymmetric pan-flavivirus amplification. Then, we demonstrated its ability to detect dengue virus with a limit of detection of 100 TCID50/mL. This magnetic field-enhanced agglutination assay is an endpoint readout, which benefits from the advantages of using nanoparticles that result in particular from a very reduced duration of the test; in our case it lasts less than 5 min. This approach provides a solution to develop new generation platforms for molecular diagnostics.

A peptide nucleic acid-regulated fluorescence resonance energy transfer DNA assay based on the use of carbon dots and gold nanoparticles.

A convenient fluorometric method was developed for specific determination of DNA based on peptide nuclei acid (PNA)-regulated fluorescence resonance energy transfer (FRET) between carbon dots (CDs) and gold nanoparticles (AuNPs). In this system, CDs that display lake blue fluorescence with excitation/emission maxima at 345/445nm were used as fluorometric reporter, while AuNPs were used as fluorescence nanoquencher. A neutral PNA probe, which is designed to recognize the target DNA, was used as a coagulant to control the dispersion and aggregation of AuNPs. Without DNA, PNA can induce immediate AuNP aggregation, thus leading to the recovery of the FRET-quenched fluorescence emission of CDs. However, the addition of the complementary target DNA can protect AuNPs from being aggregated due to the formation of DNA/PNA complexes, which subsequently produces a high fluorescence quenching efficiency of CDs by dispersed AuNPs. Under optimized conditions, quantitative evaluation of DNA was achieved in a linear range of 5-100nM with adetection limit of 0.21nM. This method exhibited an excellent specificity towards fully matched DNA. In addition, the application of this assay for sensitive determination of DNA in cell lysate demonstrates its potential for bioanalysis and biodetection. Graphical abstract A simple fluorometric biosensor for specific detection of DNA was developed based on peptide nuclei acid (PNA)-regulated fluorescence resonance energy transfer (FRET) between carbon dots (CDs) and gold nanoparticles (AuNPs).

Evaluation of serum-based real-time PCR to detect Schistosoma mansoni infection before and after treatment

BackgroundTo detect acute schistosomiasis, low-intensity infections, or to verify the success of treatment with praziquantel, highly sensitive test methods are required. The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction (PCR) in an endemic area before and after treatment.MethodsThe study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70 years in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018. Blood, urine and stool samples were collected from each participant to diagnose Schistosoma mansoni infection before and two times after treatment with praziquantel using serum- and urine based real-time PCR, point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic test and the microscopic Kato-Katz (KK) method. Kappa coefficient (κ) was used to estimate the agreement between these diagnostic tests compared to a combined “gold standard” of positive results by serum-based real-time PCR and/or positive egg counts determined by KK. Kendall’s Tau rank correlation was used to examine the relationship between cycle threshold (Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points.ResultsBy using the combined “gold standard” of the parasitological Kato-Katz test and/or serum-based real-time PCR, a S. mansoni prevalence of 77.1% could be determined at baseline. In terms of sensitivity, serum-based real-time PCR (96.3%) and POC-CCA assay (77.8%) showed the highest results. The detection of DNA from urine samples showed the lowest sensitivity (33.3%). Treatment with praziquantel resulted in a significantly reduced prevalence of S. mansoni. No infection could be detected by Kato-Katz, with the POC-CCA test only 33.3%. The analysis of the median Ct values over time (which were determined by the serum-based real-time PCR) showed that the Ct decreases significantly shortly after treatment (from 30.3 to 28) and increases above baseline level (34.9) three months later.ConclusionsThe data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy, in contrast to the use of urine as sample material for S. mansoni DNA detection. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.

Graphene based field-effect transistor biosensors functionalized using gas-phase synthesized gold nanoparticles

Research has focused on graphene for developing the next generation of label-free biosensors, capable of highly sensitive and specific detection of DNA or other biomolecules. The binding of charged analytes to the one-atom thick layer of graphene can greatly affect its electronic properties. However, graphene is highly chemically inert, thus surface functionalization through chemical treatment is typically necessary to immobilize receptors of the target biological analyte on the graphene. In this work, we use gas-phase synthesized gold nanoparticles (Au NPs) to functionalize and bind a DNA aptamer to the graphene surface. The graphene is employed in a liquid gated field-effect transistor (FET) configuration to detect the hybridization of the complementary DNA strand, as well as the protein streptavidin, at attomolar level (aM, 10−18 mol L−1). The sensor shows a high dynamic detecting range from aM to picomolar (pM) levels (10-18 to 10-12 mol L−1), can discriminate between a complementary strand and a single nucleotide polymorphism (SNP) containing strand, and achieves a detection limit as low as 15 aM. The high detection limit suggests that decorating biosensors with Au NPs synthesized from magnetron sputtering inert gas condensing technique is a promising method for biosensor functionalization, particularly for larger-area sensors that employ two-dimensional materials such as graphene.

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2=0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system

ObjectivesUndetectable or low-level hepatitis B virus (HBV) DNA and drug resistance mutations in patients may increase the risk of HBV transmission or cause active viral replication and other clinical problems. Here, we established a highly sensitive and practical method for HBV and drug resistance detection using a polymerase chain reaction (PCR) -based CRISPR-Cas13a detection system (referred to as PCR-CRISPR) and evaluated its detection capability using clinical samples. MethodsSpecific CRISPR RNAs (crRNAs) are designed for HBV DNA detection and YMDD (tyrosine-methionine-aspartate-aspartate) variant identification. The HBV DNA was detected in 312 serum samples for HBV diagnosis using quantification PCR (qPCR) and PCR-CRISPR. Additionally, 424 serum samples for YMDD testing were detected by qPCR, direct sequencing, and our assay. ResultsUsing PCR-CRISPR, one copy per test of HBV DNA was detected with HBV-1 crRNA in 15 min after PCR amplification. Consistent results with qPCR were observed for 302 samples, while the remaining 10 samples with low-level HBV DNA were detectable by PCR-CRISPR and droplet digital PCR but not by qPCR. PCR-CRISPR diagnosed all 412 drug-resistant samples detected by the YMDD detection qPCR kit and direct sequencing, as well as the other 12 drug-resistant samples with low-level HBV DNA undetectable by qPCR and direct sequencing. ConclusionsWe developed a novel PCR-CRISPR method for highly sensitive and specific detection of HBV DNA and drug resistance mutations. One copy per test for HBV DNA and YMDD drug resistance mutations could be detected. This method has wide application prospects for the early detection of HBV infection, drug resistance monitoring and treatment guidance.

Update on the major imported protozoan infections in travelers and migrants.

Globalization has contributed to the emergence of specific parasitic diseases in novel geographical areas, and in these regions, these infections in travelers and immigrants may cause a considerable burden of disease. Timely diagnosis and treatment of protozoan infections to decrease mortality and prevent associated complications are essential. In this respect, the increased availability of specific DNA-detection procedures has improved the diagnosis of many imported parasitic infections. Travelers and immigrants with associated comorbidities or immunosuppression may pose a special challenge regarding management. An updated review of the main protozoan infections in mobile populations (malaria, Chagas disease, leishmaniasis, enteric protozoan infections) is provided, focusing on the changing epidemiology of these diseases, recent developments in diagnosis and management and the possibility of local transmission of imported infections.

Amplified Analysis of DNA or Proteins by TdT-generated DNAzyme.

Sensitive and specific detection of nucleic acids or proteins, which act as biomarkers, is of great importance in disease diagnosis. By combing the concept and operation of an endonuclease-assisted target-responsive amplification method and peroxidase-mimic DNAzyme generated by terminal deoxynucleotidyl transferase (TdT), a novel and facile colorimetric biosensor was developed for DNA and protein. Target DNA and thrombin were chosen as representative biomolecules. The production of cleavage fragments can only be triggered by specific target binding and the following nicking process, which do not occur spontaneously. In the signal collection part, numerous guanine-rich DNA were produced through the prolongation of cleavage fragments by TdT and formed highly effective DNAzyme with hemin. In this novel amplification method, we succeeded in realizing sensitive and specific detection of target DNA and thrombin. Under optimal conditions, target DNA can be detected as low as 1 pM, and thrombin with a detection limit of 100 pM. The method also proves the potential versatility and feasibility of TdT-generated DNAzyme in various bio-analyses.

Absence of Viable Toxoplasma gondii in Artisanal Raw-Milk Ewe Cheese Derived from Naturally Infected Animals.

The presence of viable Toxoplasma gondii was investigated in artisanal cheeses made from milk of naturally infected ewes. Ewe milk was analyzed beforehand for the presence and vitality of T. gondii by loop-mediated isothermal amplification (LAMP) and reverse-transcriptase PCR (RT-PCR), respectively. Cheeses were prepared from raw milk following a traditional cheesemaking process. The cheese obtained from T. gondii-positive milk was analyzed by LAMP to detect Toxoplasma DNA-positive samples. RT-PCR was then carried out to assess the viability of the parasites in T. gondii-positive milk samples and fresh cheese, after 5 and 15 days of ripening. Physical-chemical parameters of cheeses were also investigated. All cheese samples derived from T. gondii-positive milk were positive according to LAMP, at both 5 and 15 days of ripening, while none of the samples were positive according to RT-PCR. Thus, while the presence of the parasite was demonstrated by the detection of specific DNA, the absence of detectable T. gondii RNA supports the hypothesis that changes in the chemical and physical characteristics occurring during the cheesemaking process and ripening period, could be sufficient to inactivate viable T. gondii in milk, minimizing the risk of human infection through consumption of raw sheep milk cheese.

Biomimetic modification of silica nanoparticles for highly sensitive and ultrafast detection of DNA and Ag+ ions

Highly sensitive and selective detection of specific DNA or metal ions based on sensor platform of dye-labeled single-stranded DNA (named as ssDNA) and functional materials has attracted great research interest over the past few decades. Herein, we reported for the first time that polydopamine (PDA) modified SiO2 nanoparticles (PDA-co-SiO2 NPs) could be severed as effective fluorescent quenchers for fabrication of fluorescent DNA sensor systems for detection of functional nuclear acids and Ag+. To prepare PDA-co-SiO2 NPs, we just need mix SiO2 nanoparticles with dopamine in aqueous solution under rather mild experimental conditions. These PDA-co-SiO2 NPs-based sensor systems possess excellent sensitivity and selectivity, fast response for detection of target ssDNA and Ag+ based on different fluorescence quenching effect of PDA-co-SiO2 NPs towards ssDNA and dsDNA. Furthermore, the high fluorescence quenching ability of PDA-co-SiO2 NPs allows quantitative detection of target ssDNA with a low detection limit of 1 nM. More importantly, this method would also be easily extended for preparation of many other PDA-based materials for fabrication of fluorescent DNA sensors owing to the universal attachment of PDA with various materials and surfaces. Therefore, this work will open up a new direction for analytical detection of PDA-based functional materials.