Sensitive DNA Detection Research Articles

A new strategy based on a cascade amplification strategy biosensor for on-site eDNA detection and outbreak warning of crown-of-thorns starfish

Population outbreaks of the crown-of-thorns starfish (COTS) seriously threaten the sustainability of coral reef ecosystems. However, traditional ecological monitoring techniques cannot provide early warning before the outbreaks, thus preventing timely intervention. Therefore, there is an urgent need for a more accurate and faster technology to predict the outbreaks of COTS. In this work, we developed an electrochemical biosensor based on a programmed catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cyclic amplification strategy for sensitive and selective detection of COTS environmental DNA (eDNA) in water bodies. This biosensor exhibited excellent electrochemical characteristics, including a low limit of detection (LOD = 18.4 fM), low limit of quantification (LOQ = 41.1 fM), and wide linear range (50 fM – 10 nM). The biosensing technology successfully allowed the detection of COTS eDNA in the aquarium environment, and the results also demonstrated a significant correlation between eDNA concentration and COTS number (r = 0.990; P < 0.001). The reliability and accuracy of the biosensor results have been further validated through comparison with digital droplet PCR (ddPCR). Moreover, the applicability and accuracy of the biosensor were reconfirmed in field tests at the COTS outbreak site in the South China Sea, which has shown potential application in dynamically monitoring the larvae before the COTS outbreak. Therefore, this efficient electrochemical biosensing technology offers a new solution for on-site monitoring and early warning of the COTS outbreak.

Abstract 2424: Detection of prostate cancer in urine by fragment-level DNA methylation analysis

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (&amp;lt;1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) &amp;gt; 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (&amp;gt;6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2424.

A universal molecular control for DNA, mRNA and protein expression

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Analytical performances of a point-of-care loop-mediated isothermal amplification assay to detect Group B Streptococcus in intrapartum pregnant women living in the Democratic Republic of the Congo

ObjectivesGroup B Streptococcus (GBS) is the leading infectious cause of stillbirth and neonatal morbidity and mortality in sub-Saharan Africa. MethodsVaginal and rectovaginal swab samples were obtained from 274 intrapartum pregnant women in the Democratic Republic of the Congo to be analyzed for GBS DNA detection in parallel by the point-of-care BIOSYNEX AMPLIFLASH® GBS assay (Biosynex SA, Illkirch-Graffenstaden, France) and by reference quantitative polymerase chain reaction (qPCR). ResultsRectovaginal swabbing, nearly two-fold more positive for GBS than vaginal swabbing alone, showed a high prevalence of GBS DNA positivity in 20.1% of eligible intrapartum pregnant women. In the event of significant bacterial carriage (i.e., cycle threshold ≤33 by reference qPCR), the AMPLIFLASH® GBS assay with rectovaginal swabbing showed high sensitivity (98.1%) and specificity (100.0%) for GBS DNA detection, with excellent concordance, reliability, and accuracy with the reference qPCR, and positive predictive values and negative predictive values above 99.0%. ConclusionsThe study demonstrates a high rate of female rectogenital GBS colonization in pregnant Congolese women. The AMPLIFLASH® GBS assay harbored excellent analytical performances in the field, which makes it suitable to be used as point-of-care molecular assay in various hospital and non-hospital settings where rapid diagnosis of GBS is necessary.

Bioluminescent Intercalating Dyes for Ratiometric Nucleic Acid Detection.

Rapid and sensitive DNA detection methods that can be conducted at the point of need may aid in disease diagnosis and monitoring. However, translation of current assays has proven challenging, as they typically require specialized equipment or probe-specific modifications for every new target DNA. Here, we present Luminescent Multivalent Intercalating Dye (LUMID), off-the-shelf bioluminescent sensors consisting of intercalating dyes conjugated to a NanoLuc luciferase, which allow for nonspecific detection of double-stranded DNA through a blue-to-green color change. Through the incorporation of multiple, tandem-arranged dyes separated by positively charged linkers, DNA-binding affinities were improved by over 2 orders of magnitude, detecting nanomolar DNA concentrations with an 8-fold change in green/blue ratio. We show that LUMID is easily combined with loop-mediated isothermal amplification (LAMP), enabling sequence-specific detection of viral DNA with attomolar sensitivity and a smartphone-based readout. With LUMID, we have thus developed a tool for simple and sensitive DNA detection that is particularly attractive for point-of-need applications.

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries.

The harmful algal bloom (HAB) species Pseudo-nitzschia multiseries is widely distributed worldwide and is known to produce the neurotoxin domoic acid, which harms marine wildlife and humans. Early detection and preventative measures are more critical than late management. However, the major challenge related to early detection is the accurate and sensitive detection of microalgae present in low abundance. Therefore, developing a sensitive and specific method that can rapidly detect P. multiseries is critical for expediting the monitoring and prediction of HABs. In this study, a novel assay method, recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD), is first developed for the detection of P. multiseries. To obtain the best test results, several important factors that affected the amplification effect were optimized. The internal transcribed spacer sequence of the nuclear ribosomal DNA from P. multiseries was selected as the target region. The results showed that the optimal amplification temperature and time for the recombinase polymerase amplification (RPA) of P. multiseries were 37 °C and 15 min. The RPA products could be visualized directly using the lateral flow dipstick after only 3 min. The RPA-LFD assay sensitivity for detection of recombinant plasmid DNA (1.9 × 100 pg/μL) was 100 times more sensitive than that of RPA, and the RPA-LFD assay sensitivity for detection of genomic DNA (2.0 × 102 pg/μL) was 10 times more sensitive than that of RPA. Its feasibility in the detection of environmental samples was also verified. In conclusion, these results indicated that the RPA-LFD detection of P. multiseries that was established in this study has high efficiency, sensitivity, specificity, and practicability. Management measures made based on information gained from early detection methods may be able to prevent certain blooms. The use of a highly sensitive approach for early warning detection of P. multiseries is essential to alleviate the harmful impacts of HABs on the environment, aquaculture, and human health.

Fluorogenic RNA aptamer output sensors via transcription activated by recombinase polymerase amplification for nucleic acid testing

Sensitive, simple, and rapid nucleic acid testing used for pathogen detection are crucial for preventing outbreaks and transmission of infectious diseases. In this study, we present a fluorogenic RNA aptamer Output Sensors via transcription Activated by Recombinase polymerase amplification (RPA), termed ROSAR, for highly sensitive detection of DNA and RNA from pathogenic bacteria or viruses in clinical samples. The promoter primer and reporter primer facilitate the integration of the transcriptional promoter with the fluorogenic RNA aptamer transcription templates through the RPA of target DNA. Subsequently, the amplification products are transcribed by T7 RNA polymerase, producing RNA aptamer that binds to a fluorogenic dye. The assay, which can be completed within 50 min and can allow a limit of detection of level aM nucleic acid concentration, owning to the dual amplification effects of RPA and in vitro transcription. Furthermore, we develop a paper-based ROSAR by freeze-drying the reaction components, which is compatible with visual detection based on mobile phone fluorescent reader and suitable for point-of-care (POC) applications. The assay can detect various viral and bacterial nucleic acids, making it a highly promising tool for large-scale epidemic screening and rapid POC testing in home healthcare settings.

Nanoplate-based digital PCR for highly sensitive pork DNA detection targeting multi-copy nuclear and mitochondrial genes

The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a ‘rain effect’ were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive ‘rain effect’ was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no ‘rain effect’. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.

One-Pot CRISPR-Cas12a-Based Viral DNA Detection via HRP-Enriched Extended ssDNA-Modified Au@Fe3O4 Nanoparticles.

In the context of virus outbreaks, the need for early and accurate diagnosis has become increasingly urgent. In addition to being crucial for effective disease control, timely and precise detection of viral infections is also necessary for the implementation of essential public health measures, especially during pandemics. Among these measures, point-of-care testing (POCT) stands out as a powerful approach with the potential to revolutionize the landscape of viral diagnosis. In this study, we developed a one-pot clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based viral DNA detection system tailored for POCT; this method utilizes multi-enzyme-modified Au@Fe3O4 nanoparticles. As an alternative to nucleic acid amplification, our method uses single-stranded DNA elongation to facilitate multi-enzyme modification; this guarantees heightened sensitivity and expedites the diagnostic process. We achieved a satisfactory limit of detection of 0.25 nM, demonstrating the remarkable sensitivity of the method without the need for sophisticated equipment. The incorporation of Au@Fe3O4 magnetic nanoparticles facilitates sample separation, further streamlining the workflow and reinforcing the simplicity of our method. This integrated approach offers a practical solution for sensitive viral DNA detection in POCT scenarios, advancing the field of rapid and accurate diagnostics.

MOF-mediated dual energy transfer nanoprobe integrated with exonuclease III amplification strategy for highly sensitive detection of DNA.

Accurate quantitative detection of DNA is an advanced strategy in various fields (such as disease diagnosis and environmental monitoring), but the classical DNA detection method usually suffers from low sensitivity, expensive thermal cyclers, or strict annealing conditions. Herein, a MOF-ERA platform for ultrasensitive HBV-DNA detection is constructed by integrating metal-organic framework (MOF)-mediated double energy transfer nanoprobe with exonuclease III (Exo III)-assisted target recycling amplification. The proposed double energy transfer containing a donor and two receptors is simply composed of MOFs (UiO-66-NH2, a well-studied MOF) modified with a signal probe formed by the hybridization of carboxyuorescein (FAM)-labeled DNA (FDNA) and black hole quencher (BHQ1)-terminated DNA (QDNA), resulting in low fluorescence signal. After the addition of HBV-DNA, Exo III degradation to FDNA is activated, leading to the liberation of the numerous FAM molecules, followed by the generation of a significant fluorescence signal owing to the negligible binding of MOFs with free FAM molecules. The results certify that the MOF-ERA platform can be successfully used to assay HBV-DNA in the range of 1.0-25.0 nM with a detection limit of 97.2 pM, which is lower than that without BHQ1 or Exo III. The proposed method with the superiorities of low background signal and high selectivity holds promise for early disease diagnosis and clinical biomedicine applications.

Ratiometric Electrochemiluminescence of Zirconium Metal-Organic Framework as a Single Luminophore for Sensitive Detection of HPV-16 DNA.

This study developed a new zirconium metal-organic framework (MOF) luminophore named Zr-DPA@TCPP with dual-emission electrochemiluminescence (ECL) characteristics at a resolved potential. First, Zr-DPA@TCPP with a core-shell structure was effectively synthesized through the self-assembly of 9,10-di(p-carboxyphenyl)anthracene (DPA) and 5,10,15,20-tetra(4-carboxyphenyl)porphyrin (TCPP) as the respective organic ligands and the Zr cluster as the metal node. The reasonable integration of the two organic ligands DPA and TCPP with ECL properties into a single monomer, Zr-DPA@TCPP, successfully exhibited synchronous anodic and cathodic ECL signals. Besides, due to the impressively unique property of ferrocene (Fc), which can quench the anodic ECL but cannot affect the cathodic ECL signal, the ratiometric ECL biosensor was cleverly designed by using the cathode signal as an internal reference. Thus, combined with DNA recycle amplification reactions, the ECL biosensor realized sensitive ratiometric detection of HPV-16 DNA with the linear range of 1 fM-100 pM and the limit of detection (LOD) of 596 aM. The distinctive dual-emission properties of Zr-DPA@TCPP provided a new idea for the development of ECL luminophores and opened up an innovative avenue of fabricating the ratiometric ECL platform.

LAMP‐based detection of Monilinia fructigena, Monilinia polystroma and Monilinia fructicola in latently infected apple fruit

AbstractBrown rot in apples is caused by fungi belonging to the genus Monilinia and occurs annually in orchards with varying severity. The symptoms of the disease on apple fruits may occur during harvest and can also develop much later in storage. Therefore, from a practical point of view, it is very important to be able to test a sample of asymptomatic apples before putting them in storage or sending them to distant markets. Thus, there is a need to develop a quick and sensitive method of detecting disease causal agents in asymptomatically infected fruit. For this purpose, a procedure for DNA isolation from the skin of apples was developed, and a set of primers for the loop‐mediated isothermal amplification (LAMP) reaction was designed based on a fragment of the hsp60 gene encoding heat shock protein 60 of Monilinia fungi that are pathogenic to apple: M. fructigena, M. fructicola and M. polystroma. The method was validated by reactions with DNA from infected apple fruit exhibiting brown rot symptoms and DNA from infected apple fruit without visible brown rot symptoms (latent infection). To increase the sensitivity of detection before the LAMP reaction, preamplification of the target DNA was carried out. The sensitivity of detection in the LAMP assay for the DNA of all three tested species of fungi was 2 pg. The DNA preamplification significantly increased the sensitivity of Monilinia DNA detection to 0.1 pg. This study shows that the developed LAMP method can be used to detect Monilinia fungi in asymptomatically infected apples.

Highly Sensitive Detection of DNA Fragments by an Inexpensive and Simple Method Fabricated In-House Blue LED Transilluminator

Agarose gel electrophoresis is a routinely employed experimental technique for visualizing nucleic acids, typically combined with ethidium bromide or some other molecular dye staining and a UV transilluminator. However, the utilization of a UV transilluminator is restricted due to high cost and safety considerations. In this study, an inexpensive and simple in-house blue Light Emitting Diode (LED) transilluminator was developed using a high-brightness blue LED, carbon resistor, and smartphone charging cable. Its sensitivity for DNA detection was compared with conventional methods. A detection limit of 4 ng/band was confirmed by observing a 100 bp DNA fragment, which is comparable to ethidium bromide staining and UV transilluminator. Additionally, the in-house blue LED transilluminator can be attached to existing electrophoresis chambers, allowing real-time observation of electrophoresis and offering a superior alternative to conventional methods. In conclusion, the developed in-house blue LED transilluminator demonstrates high sensitivity in detecting DNA fragments and holds potential for advancing low-cost research facilities.

Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis.

Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 μl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.

A “turn-on” photoelectrochemical DNA biosensor based on charge transfer between BiVO4 and gold nanoparticles

An enhanced charge transfer between BiVO4 and Au nanoparticles (AuNPs) was utilized to design a “turn-on” photoelectrochemical (PEC) biosensor for sensitive detection of target DNA. The calcination of BiVO4 on indium tin oxide (ITO) substrate was integrated with Au nanoparticles (NPs) labelled probe DNA (pDNA-AuNPs) as photoelectrode (ITO/BiVO4/pDNA-AuNPs). The cathodic photocurrent increased due to the coupling BiVO4 with AuNPs, which acted as the blank signal of this PEC platform. The probe specifically recognized and captured the target DNA (tDNA) to form double strain DNA (dsDNA) after hybridization. The photocurrent intensity of the ITO/BiVO4/dsDNA-AuNPs photoelectrode further increased due to the enhancement of the charge transfer between BiVO4 and AuNPs through dsDNA chain. This photocurrent enhancement was linear proportional to the logarithm of tDNA concentration. Based on this strategy, the PEC assay displayed a linear range from 1 aM to 10 pM and the detection limit was as low as 0.2 aM (S/N = 3). It provides a promising platform for ultrasensitive target DNA detection, and thus shows a great potential in genetic disease or cancer diagnostics and infectious pathogen detection.

Rapid DNA extraction and colorimetric amplicon visualisation speed up LAMP-based detection of soybean allergen in foods

AbstractDetection of allergens in foods, including soybean, is relevant for food labelling requirements. Moreover, allergen-specific methods may allow standardisation of allergens in food matrices for use in food challenges as allergy diagnostic approaches. Rapid methods are preferred for screening and along the manufacturing line. Previously, we demonstrated sensitive and specific detection of soybean DNA by combining loop-mediated isothermal amplification (LAMP) and lateral flow device (LFD)-like visualisation. However, lengthy DNA extraction and potential contamination of subsequent by previous LAMP reactions from unclosed LFD may impact its use as a rapid and robust method. Here, we developed a rapid protocol for DNA extraction. Moreover, we identified phenol red for distinct visualisation of positive reactions in permanently closed reaction tubes. The optimised method was validated using complex foods (boiled sausage, instant soup, and chocolate) with known amounts of soybean. Further, its applicability was shown in 12 processed retail foods. Results were verified by orthogonal qPCR. The enhanced LAMP method allowed detection at or below 10 mg soybean per kg processed food. The method provides rapid and easy-to-use screening without the need for detection equipment. Hence, it may serve to verify the presence of soybean ingredients and support a risk-based precautionary labelling of non-ingredient soybean in compound foods. Also, as determination of clinical reaction thresholds before and after allergen immunotherapy (AIT) is both inclusion and exclusion criterion for clinical trials and success parameter of AIT, the method may allow verification of calculable soybean content in provocation meals and thus a standardised administration for threshold determination before and after AIT.

Au/NG/SWCNTs/FTO-glass modified electrode based electrochemical biosensor for DNA detection

An easy, quick, and efficient detection of Multidrug Resistance (MDR) of cancer cells at the clinical level is the need of the hour for the effective treatment of leukemia patients. In this study, we reported carbon nanomaterials-based biosensors based on Au nanoparticles (Au-NPs) decorated on Nitrogen-doped Graphene (NG) and Single-Walled Carbon Nanotubes (SWCNTs) for effective, efficient, and sensitive detection of affected DNA. Three Au/NG/SWCNT-based Modified Electrode (ME) were made containing 2, 4, and 6 mg SWCNTs. The thiol group attached at the 5′end was immobilized at ME due to a strong bond between the Au-NPs and thiol group. NG/SWCNTs enhanced the performance of the composite due to its better sensitivity and fast electron transport. Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV), and Differential Potential Voltammetry (DPV) analysis were performed to monitor the performance of the sensor after the hybridization of target DNA onto the capture probe on the modified electrode surface. Different target DNA sequences were used to check the selectivity and sensitivity of the fabricated biosensor. The selectivity performance of prepared DNA sensors was also proved to be good shown by DPV results. The findings of this study provide insights for developing new highly sensitive, selective, and cost-effective portable biosensors for MDR gene detection, which will enable the early diagnosis of cancer more effective at the clinical level. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity.

Nanomechanoelectrical approach to highly sensitive and specific label-free DNA detection

Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical methods, which solely rely on measuring electrical signals in transducers during probe-target DNA hybridization, are prone to nonspecific electrostatic and electrochemical interactions, subsequently limiting their specificity and detection limit. Here, we demonstrate a nanomechanoelectrical approach that delivers ultra-robust specificity and a 100-fold improvement in detection limit. We drive nanostructural DNA strands tethered to a graphene transistor to oscillate in an alternating electric field and show that the transistor-current spectra are characteristic and indicative of DNA hybridization. We find that the inherent difference in pliability between unpaired and paired DNA strands leads to the spectral characteristics with minimal influence from nonspecific electrostatic and electrochemical interactions, resulting in high selectivity and sensitivity. Our results highlight the potential of high-performance DNA analysis based on miniaturized all-electronic settings.

CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood

BackgroundToxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.MethodsThis study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.ResultsThe results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi).ConclusionsThis study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.Graphical abstract