Probe Amplification Research Articles

P-2139. Evaluation of a Multiplex Real-time PCR for the Detection and Differentiation of Sporothrix schenckii and Sporothrix brasiliensis

Abstract Background Sporothrix spp is a thermally dimorphic fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases.Figure 1.Limit of detection and genomic DNA standard curve of S. schenckii and S. brasiliensis Methods A recently described Real-time PCR assay targeting the β-tubulin gene to detect and differentiate two related species, S. schenckii and S. brasiliensis, was evaluated and adapted by adding lambda primers and probe for internal control amplification. Isolates, fresh tissues and suspected sporotrichosis Formalin-Fixed, Paraffin Embedded (FFPE) tissues from humans and animals were received in the Mycotic Diseases Branch laboratory at the U.S. Centers for Disease Control and Prevention (CDC) for the routine fungal identification by conventional PCR and sequencing, using the primers D1/D2, ITS5/ITS4 and ITS3/ITS4. Ten-fold serial dilutions from 1 ng/μl to 1 fg/μl of DNA were tested with 20 replicates to establish limit of detection (LOD), and DNA from four fresh tissues extracted on three different days was used to assess reproducibility.Table 1.Ct values and coefficient of variation (CV) of the reproducibility test using fresh tissues on three different days Results The assay was tested with 55 S. brasiliensis, 19 S. schenckii, and 85 isolates from other clinically relevant fungi, showing 100% concordance with sequencing methods. The test was also able to amplify S. schenckii DNA from 10 fresh tissues, which were previously confirmed by culture and sequencing as S. schenckii (100% sensitivity), with Ct values ranging from 19 to 38. Among 9 FFPE samples, S. schenckii was amplified in 6 samples (67% sensitivity) with Ct values ranging from 29 to 38. LOD was 1pg/μl of DNA. Results of the 20 replicates showed that at this concentration all the DNAs had a positive signal for both targets (fig 1). For reproducibility, the results showed consistent Ct values over the 3 days of the test with the four fresh tissue samples with a coefficient of variation (CV) of 4% (table 1). Conclusion This multiplex Real-time PCR assay successfully detected DNA from the S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this molecular assay performs well and could be a helpful molecular tool to support rapid identification and differentiation at species level from cultures and primary human and animal specimens. Disclosures All Authors: No reported disclosures

Visible and rapid detection of feline chaphamaparvovirus using multienzyme isothermal rapid amplification and lateral flow dipstick assay

Feline chaphamaparvovirus (FeChPV) is a novel parvovirus previously reported in Canadian cats and Chinese dogs with diarrhea in 2019 and 2020, respectively. Herein, we aimed to establish a simple detection method for FeChPV in field clinics. The primers and probes for the multienzyme isothermal rapid amplification and lateral flow dipstick (MIRA-LFD) assay were designed to target the conserved regions of the FeChPV genome and determine the optimal reaction temperature and time. Without relying on precision instruments, FeChPV detection using the MIRA-LFD assay was completed within 20 min at 37°C, without any cross-reaction with other reference viruses. The newly established MIRA-LFD assay had a detection limit of 32.3 copies/μL, which was 10-fold lower than that of the nested polymerase chain reaction (PCR) assay. Furthermore, the MIRA-LFD assay detected 29 FeChPV-positive samples among 417 cats with diarrhea, providing a slightly higher positivity rate than the nested PCR assay. These results indicate that the newly developed MIRA-LFD assay for FeChPV detection is an efficient, economical, reliable, and simple method that can help in the early prevention and control of FeChPV infection.

Nanozyme-enhanced tyramine signal amplification probe for preamplification-free myocarditis-related miRNAs detection

Signal amplification-based sensors enable sensing of low-abundance miRNA markers associated with myocarditis diseases in biological fluids. However, most miRNA sensing protocols usually adopt pre-amplification to achieve the required sensitivity, which brings complexity and non-specificity to the experiment. Here, we showcase a strategy that combines tyramine signal amplification (TSA) with nanozyme catalysis, which allows for the quantitative and colorimetric readout of miRNA with high sensitivity and specificity via TSA-mediated infinite assembly of nanozymes (nano TSA). We demonstrated the analysis of miRNA in biological samples (blood, cell, and heart). Furthermore, we utilize this efficient miRNA-detecting technique enables to the identification of acute myocarditis patients. We expected that nanozyme would serve as a commonly applicable signal enhancement catalyst for nano TSA-based diagnostics, which will pave the way for enlarging the preamplification-free quantitative detection of trace targets.

Accurate HER2 determination in breast cancer: a prominent COF-immobilized enzyme-enhanced electrochemical aptasensor employing 4-acetamidophenol as an efficient mediator

An effective strategy for enzyme-enhanced electrochemical detection of human epidermal growth factor receptor 2 (HER2) is proposed for breast cancer diagnosis. This strategy utilizes a three-dimensional mesoporous covalent organic framework (COF), immobilized horseradish peroxidase (HRP), and a novel redox mediator, 4-acetamidophenol (APAP). The mesoporous structure, with encapsulation effect, and good biocompatibility of COF, makes the functionalized COF an efficient carrier for HRP immobilization (HRP-Ab-AuNPs@COF). It demonstrates superior catalytic activity, stability, and electrochemical performance compared to free HRP, thus making it an ideal probe for simultaneous target recognition and signal amplification. APAP is screened from four candidate phenolic compounds based on its high formal potential (0.32 V vs. Ag/AgCl), rapid electron transfer activity (kapp = 2.80 × 105 M− 1 s− 1), excellent solubility and stability. These properties prove significantly better than the conventional mediator hydroquinone (HQ), achieving a higher signal-to-background ratio. By integrating decorated multi-walled carbon nanotubes as substrate materials, the electrochemical aptasensor achieves a low HER2 detection limit (0.418 pg mL− 1) with high specificity. This method’s selectivity surpasses that of the HQ-mediated method by 59–73%. Moreover, the aptasensor can effectively distinguish breast cancer patients and healthy individuals, as well as patients at different stages of the disease with high accuracy (AUC = 0.928). This performance exceeds traditional biomarkers CEA and CA15-3. This work paves novel avenues for innovative applications of COF-immobilized enzymes and the novel mediator APAP in electrochemical biosensing, thus holding significant promise for individualized breast cancer diagnosis and treatment.

A DNA sensor based on CbAgo effector protein and on a dual electrochemical signal amplification strategy for B19 parvovirus detection

Human parvovirus B19 is a prevalent childhood infectious virus that poses a great challenge to public health, so the detection of B19V is of great importance. In this study, a DNA sensor based on CbAgo, a Cas effector, and a dual electrochemical signal amplification strategy was developed by using a novel nanocomposite MnO2/CMK-3/g-C3N4/AgNPs for initial signal amplification, with CMK being an ordered mesoporous carbon nanomaterial. Single-walled carbon nanotubes (SWCNTs) were used as electrocatalytic probes for secondary signal amplification to detect B19 DNA. The detection process begins with polymerase chain reaction (PCR) amplification using the B19V infectious clone plasmid (pB19-M20) as a template and NS1-F/R as primers, followed by specific cleavage of B19 DNA based on the programmable cutting sites of CbAgo effector protein. This study enriches the application of Argonaute proteins in sensing and introduces a novel method to detect B19V. Under optimized conditions, the biosensor can detect B19 DNA in the range of 10−15–10−10 M, with a detection limit (LOD) of 0.2 fM. The results indicate that the developed DNA sensor holds promise for reliable and sensitive detection of B19 DNA in human serum.

Development and optimization of the qPCR-based rapid detection method for Chinese species of Sanghuangporus

Sanghuangporus, well known as ‘Sanghuang’, is a macrofungal genus accommodating 11 edible and medicinal species in China. With the prosperous development of ‘Sanghuang’ business in China, the fraudulent practice naming the products of other morphologically and phylogenetically related species as ‘Sanghuang’ happens frequently, which disturbs the market order of ‘Sanghuang’ business. To promote the healthy industrial development of Sanghuangporus and also prevent the loss of this macrofungal resource, we develop a qPCR-based rapid detection method in this study. Internal transcribed spacer sequences of Fuscoporia, Inonotus, Phellinus, Sanghuangporus and Tropicoporus were aligned to design Sanghuangporus-specific amplification primers and probe, which can specifically distinguish all 11 Chinese species of Sanghuangporus from species of other closely related genera. The concentrations of Sanghuangporus-specific primers and probe were optimized as 0.7 μmol/L and 0.5 μmol/L, respectively, where the qPCR has the minimum detectable DNA template concentration of 10−3 ng/μL. In summary, the current qPCR-based detection method was specific, sensitive and rapid for Chinese species of Sanghuangporus.

Optical genome mapping of structural variants in Parkinson’s disease-related induced pluripotent stem cells

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants, as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast, larger SVs, which may significantly influence human phenotypes, have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study, we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines, which were not identified by long-read sequencing. Additionally, various exon deletions were confirmed by OGM in the PRKN gene of iPSCs, of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs, no gene fusions, no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary, OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques, which is supportive for OGM’s future use in gene discovery and iPSC line screening.

Nucleic Acid Amplification Circuit-Based Hydrogel (NACH) Assay for One-Step Detection of Metastatic Gastric Cancer-Derived Exosomal miRNA.

Gastric cancer (GC) is recognized as the fifth most prevalent malignant tumor worldwide. It is characterized by diverse clinical symptoms, treatment responses, and prognoses. In GC prognosis, the promotion of epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, thereby triggering the dissemination of tumor cells. This study proposes a nucleic acid amplification circuit-based hydrogel (NACH) assay for identifying exosomal miRNA derived from metastatic GC. The NACH assay employs the rolling circle amplification method and targets miRNA-21, a tumor-related oncogene, and miRNA-99a, which promotes EMT. Specific amplification probes for each target are immobilized within the hydrogel, enabling a streamlined, one-step amplification reaction. The NACH assay exhibits a detection limit of 1fm for miRNA-21 and miRNA-99a, thereby enabling rapid and highly sensitive on-site detection. Performance evaluation using exosomal miRNA extracted from cell culture media, mouse plasma, and human plasma revealed fluorescence intensity patterns similar to those obtained in qRT-PCR. Furthermore, deploying a custom-developed portable fluorometer for the NACH assay allows for diagnostic performance assessment and point-of-care testing using clinical samples from GC patients. These findings emphasize the potential of the NACH assay to be used as a robust tool for the genetic diagnosis of GC based on exosome detection.

Ultrasensitive Electrochemiluminescence Biosensor with ZIF-67@MXene as an Efficient Co-Reaction Accelerator and Plasmonic Nanozyme as a Smart Signal Amplification Probe.

Exploring novel electrochemiluminescence (ECL) co-reaction accelerators to construct ultrasensitive sensing systems is a prominent focus for developing advanced ECL sensors. However, challenges still remain in finding highly efficient accelerators and understanding their promoting mechanisms. In this paper, ZIF-67@MXene nanosheet composites, with highly conductive in-plane structure and confined-stable pore/channel, are designed to act as high-efficient co-reaction accelerators and achieve a significant enhancement in the luminol-H2O2 based ECL system. Mechanism investigation suggests that hydroxyl radicals (·OH) and singlet oxygen (1O2) can be selectively and preferentially generated on ZIF-67@MXene due to the stable and efficient absorption of ·OH and 1O2, leading to a remarkable enhancement in the ECL efficiency of luminol (830%). Finally, by designing a plasmonic NH2-MIL-88@Pd nanozyme, an "on-off" switch immunosensor is constructed for the detection of prostate-specific antigen (PSA). Based on the multiple signal amplification effect, the linear detection range for PSA is expanded by three orders of magnitude. The detection limit is also improved from 1.44 × 10-11 to 9.1 × 10-13 g mL-1. This work proposes an effective method for the preparation of highly efficient co-reaction accelerators and provides a new strategy for the sensitive detection of cancer markers.

An aptasensor with colorimetric and electrochemical dual-outputs for malathion detection utilizing peroxidase-like activity of Fe-MOF

An aptasensor with dual-outputs was developed for malathion detection. Fe-MOF was synthesized to design favorable signal probes for catalytic amplification. Owing to the excellent peroxidase-like activity of Fe-MOF, the redox reaction was catalyzed to produce the dual-outputs of colorimetric and electrochemical. In this sensing strategy, malathion was captured by the aptamer on sensing interface, leading to the release of signal probe. Thanks to the catalytic amplification of Fe-MOF and the high capture effect of aptamer, the aptasensor produced a sensitive response for malathion. Based on the dual-signals of absorbance and current, the detection method for malathion was developed ranging from 10 ng/mL to 500 ng/mL. The detection limit of malathion was 5.8 ng/mL for colorimetric output and 4.6 ng/mL for electrochemical output. Furthermore, the aptasensor exhibited high specificity and good repeatability in malathion detection. Finally, the aptasensor was applied to detect malathion in fruit and vegetable samples with satisfactory recovery.

Polypyrrole deposited on the core-shell structured nitrogen-doped porous carbon@Ag-MOF for signal amplification detection of chloride ions.

An electrochemical platform for signal amplification probing chloride ions (Cl-) is constructed by the composite integrating core-shell structured nitrogen-doped porous carbon@Ag-based metal-organic frameworks (NC@Ag-MOF) with polypyrrole (PPy). It isbased on the signal of solid-state AgCl derived from Ag-MOF, sinceboth NC and PPy have good electrical conductivityand promote the electron transport capacity of solid-state AgCl. NC@Ag-MOF was firstly synthesized with NC as the scaffold and then, PPy was anchored on NC@Ag-MOF by chemical polymerization. The composite NC@Ag-MOF-PPy was utilized to modify theelectrode, which exhibited a higher peak current and lower peak potential during Ag oxidation compared with those of Ag-MOF and NC@Ag-MOF-modified electrodes. More importantly, in the coexistence of chloride (Cl-) ions in solution, theNC@Ag-MOF-PPy-modified electrode displayed a fairly stable and sharp peak of solid-state AgCl with the peak potentials gradually approaching zero, which might effectively overcome the background interference caused by electroactive substances. Theoxidation peak currents of solid-state AgCl increased linearly with the concentrationof Cl- ions in a broad range of 0.15µM-40mM and 40-250mM, with detection limits of 0.10µM and 40mM, respectively. Thepractical applicability for Cl- ions determinationwas demonstrated usinghuman serum and urinesamples. Theresults suggest that NC@Ag-MOF-PPy composite could be a promising candidate for the construction of the electrochemical sensor.

DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL−1, 1.5 copies⋅μL−1, and 0.17 copies⋅μL−1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

Janus plasmonic-aggregation induced emission nanobeads as high-performance colorimetric–fluorescent probe of immunochromatographic assay for the ultrasensitive detection of staphylococcal enterotoxin B in milk

Herein, a colorimetric–fluorescent hybrid bifunctional nanobead with Janus structure (J-cf-HBN) was synthesized via one-pot microemulsification. Oleylamine-coated AuNPs and aggregation-induced emission luminogens (AIEgens) were suggested as building blocks to obtain high-performance colorimetric–fluorescent signals. The as-prepared J-cf-HBNs were used as a signal amplification probe to construct an immunochromatographic assay (J-cf-HBNs-ICA) platform for the ultrasensitive detection of staphylococcal enterotoxin B (SEB) in milk samples. Owing to the rational spatial distribution of AuNPs and AIEgens, the J-cf-HBNs present a highly retained photoluminescence and enhanced colorimetric signals. Combined with a pair of highly affinitive anti-SEB antibodies, the J-cf-HBN-ICA platform enabled the fast naked-eye visualization and fluorescent quantitative detection of SEB in various milk matrices. Given the advantages of the dual-mode high-performance J-cf-HBNs, the proposed strip achieved a high sensitivity for SEB qualitative determination with a visual limit of detection (LOD) of 1.56 ng mL−1 and exhibited ultrasensitivity for SEB quantitative detection with a LOD of 0.09 ng mL−1, which is 139-fold lower than that of ELISA using same antibodies. In conclusion, this work provides new insights into the construction of multimode immunochromatographic methods for food safety detection in the field.

Efficient electrochemical immunosensor for detection of bovine interferon gamma‐based gold nanoparticles/chitosan composite

AbstractInterferon gamma (IFN‐γ) released by T lymphocytes, plays an important role in immune regulation and is also an important biomarker for the early diagnosis of bovine tuberculosis. In this work, an electrochemical immunosensor based on gold nanoparticles (AuNPs)/chitosan composite was developed for highly sensitive determination of bovine IFN‐γ. AuNPs/chitosan composite shows high surface area and good hydrophilicity. The bovine IFN‐γ immunosensor was prepared by immobilizing IFN‐γ antibody on AuNPs/chitosan composite‐modified glassy carbon electrode. The immunosensors were characterized by scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry (CV). Using horseradish peroxidase (HRP)‐labeled IFN‐γ antibody as amplification probe, the concentration of bovine IFN‐γ can be monitored via the increasing current signal, which can be attributed to the specific immune binding between bovine IFN‐γ antigen and HRP‐labeled IFN‐γ antibody, as well as the efficient catalysis reaction between thionine and HRP with the presence of H2O2. The developed electrochemical immunosensor exhibited a wide linear range of 0.01 to 30 ng/mL, and low detection limit of 0.003 ng/mL with good specificity and stability. This study provides a simple and promising method for diagnosis of bovine tuberculosis, which can be expanded to determine other biomarkers.

Nucleic acid detection with single-base specificity integrating isothermal amplification and light-up aptamer probes.

We report a novel platform for label-free nucleic acid detection using isothermal amplification and light-up aptamer probes. This assay converts double-stranded amplicons into single-stranded targets to enable sequence-specific hybridization with split dapoxyl aptamer probes, offering attomolar sensitivity and single-base specificity.

A novel β-cyclodextrin-assisted enhancement strategy for portable and sensitive detection of miR-21 in human serum.

Benefiting from our discovery that β-cyclodextrin (β-CD) could enhance the catalytic activity of invertase through hydrogen bonding to improve detection sensitivity, a highly sensitive and convenient biosensor for the detection of miR-21 was proposed, which is based on the simplicity of reading signals from a personal glucose meter (PGM), combined with self-assembled signal amplification probes and the performance of β-CD as an enhancer. In the presence of miR-21, magnetic nanoparticle coupled capture DNA (MNPs-cDNA) could capture it and then connect assist DNA/H1-invertase (aDNA/H1) and self-assembled signal amplification probes (H1/H2) in turn. As a result, a "super sandwich" structure was formed. The invertase on MNPs-cDNA could catalyze the hydrolysis of sucrose to glucose and this catalytic process could be enhanced by β-CD. The PGM signal exhibited a linear correlation with miR-21 concentration within the range of 25 pmol L-1 to 3 nmol L-1, and the detection limit was as low as 5 pmol L-1 with high specificity. Moreover, the recoveries were 103.82-124.65% and RSD was 2.59-6.43%. Furthermore, the biosensor was validated for the detection of miR-21 in serum, and the results showed that miR-21 levels in serum samples from patients with Diffuse Large B-Cell Lymphoma (DLBCL) (n = 12) were significantly higher than those from healthy controls (n = 12) (P < 0.001). Therefore, the ingenious combination of PGM-based signal reading, self-assembled signal amplification probes and β-CD as an enhancer successfully constructed a convenient, sensitive and specific biosensing method, which is expected to be applied to clinical diagnosis.

Imaging and Enumeration of Fluorescence in situ Hybridization Amplification Probe using Structured-Light Illumination Super-Resolution Microscopy

Imaging and Enumeration of Fluorescence in situ Hybridization Amplification Probe using Structured-Light Illumination Super-Resolution Microscopy

Dual-channel MIRECL portable devices with impedance effect coupled smartphone and machine learning system for tyramine identification and quantification.

We designed a novel, portable, and visual dual-potential molecularly imprinted ratiometric electrochemiluminescence (MIRECL) sensor for tyramine (TYM) detection based on smartphone and deep learning-assisted optical devices. Molecularly imprinted polymer-Ce2Sn2O7 (MIP-Ce2Sn2O7) layers were fabricated by in-situ electropolymerization method as the capture and signal amplification probe. Oxygen vacancies in Ce2Sn2O7 not only enhance the electrochemical redox capability but also accelerate the energy transfer, thereby enhancing the luminescence of cathode ECL. Under optimal conditions, the ECL signals of MIP-Ce2Sn2O7 at the cathode and the anode response of Ru(bpy)32+ was reduced, thus a wide linear range from 0.01μM to 1000μM with the detection limit as low as 0.005μM. Interestingly, combined with an artificial intelligence image recognition algorithm and the principle of optical signal reading by smartphone, the developed MIRECL sensor has been applied to the portable and visual determination of TYM in aquatic samples, and its practicability has been satisfactorily verified.

Development of a real-time enzymatic recombinase amplification assay for rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp Penaeus vannamei

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is classified as a reportable crustacean disease by the World Organisation for Animal Health (WOAH), which causes poor growth in Penaeus vannamei. According to genome sequence alignment analysis, enzymatic recombinase amplification (ERA) primers and probe were designed based on the ORF1 region of IHHNV, and a real-time ERA assay for IHHNV detection (IHHNV-ERA) was established. The experimental results show that IHHNV-F2/IHHNV-R2 and IHHNV-Probe can effectively amplify the target gene, and the sensitivity is 1.4 × 101 copies/μL within 14.97 ± 0.19 min, while the qPCR using primers 309F/309R could reach the detection limit of 1.4 × 101 copies/μL within 21.76 ± 0.63 min, and the sensitivity results of one-step PCR could be as low as 1.4 copies/μL with expense of time and false positives. The IHHNV-ERA system can effectively amplify the target gene at 42 ℃ within 20 min, and has no cross-reaction with white spot syndrome virus (WSSV), Ecytonucleospora hepatopenaei (EHP), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), and healthy shrimp genomic DNA. Test results of practical samples showed that the detection rate of IHHNV-ERA (18/20) was better than the industrial standard qPCR assay (17/20). Compared with the existing technology, the useful results of this detection assay are: (1) get rid of the dependence on the thermal cycle instrument in the PCR process; (2) the experimental procedure is simple, time-consuming and fast; (3) the detection sensitivity is high. This study provides an ERA based detection assay for IHHNV, which can be used not only for the rapid detection of IHHNV infection, but also for the field screening of pathogens. This assay can also be applied to clinical inspection, customs detection, enterprise quality inspection and other fields, and has obvious practical application value.

Self-assembly of DNA-gold nanoaggregate for visual detection of thymidine kinase 1 (TK1) mRNA via lateral flow assay.

Nucleic acid lateral flow assay (NALFA) with gold nanoparticles (AuNPs) as colorimetric probes have been extensively adopted for point-of-care testing (POCT). However, the sensitivity of NALFA still needs to be improved. Herein, DNA-gold nanoaggregate (DNA-AuNA) was assembled as a signal amplification probe of NALFA for sensitive detection of tumor marker TK1 mRNA. Four functional oligonucleotides with complementary pairs were assembled to form DNA-AuNA that coupled more AuNPs to improve sensitivity. Thus, the limit of detection (LOD) was 0.36pM, which is lower than that of conventional AuNPs-based NALFA. Moreover, the bioassay showed good reproducibility, stability, and specificity for detecting TK1 mRNA. The detection of TK1 mRNA in human serum was also satisfactory. Therefore, DNA-AuNA-based NALFA provides a sensitive method for portable detection of TK1 mRNA.