Nucleic Acid Amplification System Research Articles

Evaluation of a nucleic acid amplification system, GENECUBE, for rapid detection of staphylococcal nuc and mecA in blood culture samples

ObjectiveThis study determined whether the GENECUBE rapid nucleic acid amplification test could directly detect nuc and mecA genes in clinical blood culture samples of Staphylococcus and various other pathogens. MethodsBetween September 2020 and December 2021, 537 blood culture samples from 192 patients with suspected bacteremia were tested using conventional assays (MicroScan WalkAway96 or VITEK 2 systems) and GENECUBE nuc and mecA assays. Isolates from samples with discrepant results between the conventional and GENECUBE assays were further evaluated using MALDI-TOF mass spectrometry, disk diffusion testing using cefoxitin, broth microdilution testing using oxacillin, and sequencing for mecA. Bacterial solutions containing a mixture of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were prepared to evaluate the limit of detection (LOD) of mecA. ResultsUsing conventional assays as the reference, the sensitivity, specificity, and positive and negative predictive values (95 % confidence interval) of GENECUBE were 100 % (96.8–100 %), 100 % (99.1–100 %), 100 % (96.8–100 %), and 100 % (99.1–100 %), respectively, for nuc detection and 100 % (96.1–100 %), 98.9 % (97.4–99.6 %), 94.9 % (88.5–98.3 %), and 100 % (99.2–100 %), respectively, for mecA detection. Sequencing analysis of five samples identified as methicillin-sensitive staphylococci using conventional assays and methicillin-resistant staphylococci using GENECUBE revealed the presence of methicillin-resistant isolates in all samples. The estimated LOD of mecA was 104 colony-forming units (CFU)/mL of MRSE with GENECUBE, compared with 105 CFU/mL with conventional assays. ConclusionThe GENECUBE assay accurately detected mecA in positive blood culture samples and had higher sensitivity than conventional assays.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

A photocontrolled one-pot isothermal amplification and CRISPR-Cas12a assay for rapid detection of SARS-CoV-2 Omicron variants.

CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensitivity of 97.3% and a specificity of 100.0% and showed a concordance of 98.3% with Sanger sequencing results.IMPORTANCEWe successfully developed one-pot recombinase polymerase amplification/CRISPR-Cas12a genotyping assay by adapting photocontrolled CRISPR-Cas technology to optimize the conditions of nucleic acid amplification and CRISPR-Cas12a-mediated detection. This innovative approach was able to quickly distinguish severe acute respiratory syndrome coronavirus 2 Omicron variants and can be readily modified for detecting any nucleic acid mutations. The assay system demonstrates excellent clinical performance, including rapid detection, user-friendly operations, and minimized risk of contamination, which highlights its promising potential as a point-of-care testing for wide applications in resource-limiting settings.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

Rapid and portable bunyavirus SFTSV RNA testing utilizing catalytic hairpin assembly coupled with lateral flow immunoassay.

Severe fever with thrombocytopenia syndrome (SFTS) is a prevalent, life-threatening, emergent infectious disease. Currently, reverse transcription-polymerase chain reaction is the gold standard for diagnosing SFTS virus (SFTSV) infection, which requires sophisticated equipment and professional personnel that are frequently unavailable in most SFTS endemic rural areas. Here, we reported a simple, rapid nucleic acid amplification system that combined the catalytic hairpin assembly (CHA) with a lateral flow immunoassay (LFIA) strip-based detection method for SFTSV detection. The detection of SFTSV RNA could be realized by generation of H1-H2 hybrid duplexes labeled with biotin and digoxin, which subsequently added to the LFIA test strips containing streptavidin conjugated with Alexa Fluor 647 as well as anti-digoxin antibodies. Our CHA-based LFIA assay offered high amplification efficiency and specificity with a detection limit of 1 aM. Crucially, this method enabled stable detection of 500 copies/mL of SFTSV within 30 min using clinical serum samples. Therefore, our CHA-based LFIA approach provided a potential useful tool to facilitate early and precise diagnosis of SFTS patients in poorly resourced SFTS endemic areas.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is an emerging and potentially fatal infectious disease prevalent in China. Here we report a simple, rapid nucleic acid amplification system, the catalytic hairpin assembly (CHA) in conjunction with a lateral flow immunoassay (LFIA) strip-based detection method for SFTS virus detection, which demonstrated high amplification efficiency and specificity with limit of detection of 1 aM. Most importantly, we also validate our CHA-based LFIA assay using the clinical serum samples, which was fully compatible with reverse transcription-PCR results. Therefore, our strategy provides a potential useful tool to facilitate early and precise diagnosis of SFTS patients especially in poorly resourced SFTS endemic areas.

UV-Assisted Hyperbranched Poly(β-amino ester) Modification of a Silica Membrane for Two-Step Microfluidic DNA Extraction from Blood.

Integrating nucleic acid extraction in amplification-based point-of-care diagnostics will be a significant feature for next-generation point-of-care virus detection devices. However, extracting DNA efficiently on a microfluidic chip poses many technological and commercialization challenges, including manual steps, multiple instruments, pretreatment processes, and the use of organic solvents (ethanol, IPA) that inhibit detection, which is not viable with routine testing such as viral load monitoring of transplant patients for post-operative care. This paper presents a microfluidic system capable of two-step DNA extraction from blood using a UV-assisted hyperbranched poly(β-amino ester) (HPAE)-modified silica membrane for cytomegalovirus (CMV) detection in a rapid and instrument-free manner without the presence of amplification inhibitors. HPAEs of varying branch ratios were synthesized, screened, and coated on a silica membrane and bonded between two layers of poly(methyl methacrylate) (PMMA) substrates. Our system could selectively extract DNA from blood with an efficiency of 94% and a lower limit viral load of 300 IU/mL in 20 min. The extracted DNA was used as the template for real-time loop-mediated isothermal amplification (LAMP)-based detection of CMV and was found to produce a fluorescent signal intensity that was comparable with commercially extracted templates. This system can be integrated easily with a nucleic acid amplification system and used for routine rapid testing of viral load in patient blood samples.

Fluidic Spatial-Confinement Scaffold Affords a Multicomponent DNA Reaction with Improved Efficiency and Accelerated Kinetics.

Enzyme-free nucleic acid amplification reactions with the capability of signal catalytic amplification have been widely used in biosensors. However, these multicomponent, multistep nucleic acid amplification systems often suffer from low reaction efficiency and kinetics. Herein, inspired by the natural cell membrane system, we utilized the red blood cell membrane as a fluidic spatial-confinement scaffold to construct a novel accelerated reaction platform. By simply modifying with cholesterol, DNA components can be efficiently integrated into the red blood cell membrane through hydrophobic interactions, which greatly increases the local concentration of DNA strands. Moreover, the fluidity of the erythrocyte membrane improves the collision efficiency of DNA components in the amplification system. Based on the increased local concentration and improved collision efficiency, the fluidic spatial-confinement scaffold significantly improved the reaction efficiency and kinetics. Taking catalytic hairpin assembly (CHA) as a model reaction, an RBC-CHA probe based on the erythrocyte membrane platform enables a more sensitive detection of miR-21 with a sensitivity that is 2 orders of magnitude higher than the free CHA probe and a fast reaction rate (about 3.3-fold). The proposed strategy provides a new idea for the construction of a novel spatial-confinement accelerated DNA reaction platform.

Intelligent Genetic Decoding System Based on Nucleic Acid Isothermal Amplification for Non-Small Cell Lung Cancer Diagnosis

Non-small cell lung cancer (NSCLC) is a major cause of cancer-related deaths around the world. Targeting the sensitized epidermal growth factor receptor (EGFR) caused by gene mutation through the tyrosine kinase inhibitor is an effective therapeutic strategy for NSCLC. Hence, the individualized therapeutic strategy has highlighted the demand for a simple, fast, and intelligent strategy for the genetic decoding of EGFR to cater to the popularization of precision medicine. In this research, a one-pot assay for EGFR identification is established by combining a loop-mediated isothermal amplification and amplification refractory mutation system. By optimizing the component and condition of the nucleic acid amplification system, a sensitive and specific distinguishability is achieved for tracing target variant (60 copies, 0.1%) identification under a strong interferential background within 40 min. Moreover, complex operation and time-consuming data processing, as well as the aerosol contamination, are avoided owing to the whole process for intelligent genetic decoding being performed in a sealed tube. As a demonstration, L858R, the primary point mutation for the sensitization of EGFR, has been accurately decoded using this assay with highly heterogeneous cancerous tissue. In addition, this method can be easily extended for other genetic information decoding using a tailor-made primer set. Thus, we propose that this straightforward strategy may serve as a promising tool for NSCLC diagnosis in clinical practice.

Validation of the Assurance® GDS for Cronobacter Tq II in Infant Formulas, Infant Cereals, Ingredients, and Environmental Samples Collaborative Study: First Action 2021.08.

The Assurance® GDS for Cronobacter Tq II assay is a nucleic acid amplification system for the qualitative detection of Cronobacter. The method uses an upfront concentration of the target organism from the enrichment by immunomagnetic separation (IMS) using the PickPen®device. The Assurance GDS for Cronobacter Tq II method was evaluated for Official Methods of AnalysisSM certification. The matrix was compared to the ISO 22964:2017: Microbiology of the Food Chain-Horizontal Method for the Detection of Cronobacter spp. standard and using an alternative confirmation procedure. The alternative method was evaluated using 10 g test portions in an unpaired study design for powdered infant formula (milk based with iron and DHA) containing probiotics. Eleven technicians from eight laboratories located within the United States and Europe participated in the collaborative study. Statistical analysis was conducted according to the probability of detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence intervals across collaborators was calculated for each level between the candidate and reference method results and between the candidate presumptive and confirmed results. Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval of 0.03 (-0.18, 0.15). The dLPOD results indicate equivalence between the candidate method and reference method for the matrix evaluated. The method also demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. There were no false negative results; the false positive rate was determined and produced a value of <2%. Based on the data generated, the method demonstrated Assurance GDS for Cronobacter Tq II assay produced acceptable interlaboratory reproducibility data and statistical analysis. The Assurance GDS for Cronobacter Tq II method is suitable for the rapid qualitative detection of Cronobacter in infant formulas, infant cereals, ingredients, and environmental samples.

Intermolecular and Intramolecular Priming Co-directed Synergistic Multi-strand Displacement Amplification Empowers Ultrasensitive Determination of microRNAs.

The construction of isothermal nucleic acid amplification systems with extremely high signal amplification abilities is of great importance for biomarker detection and disease diagnosis. Herein, for the first time, we proposed an intermolecular and intramolecular priming co-directed synergistic multi-strand displacement amplification (SM-SDA) system for microRNA (miRNA) detection. Strategically, the SM-SDA system is made of a single multi-functionalized hairpin probe (MF-HP) that is engineered with a long stem and endowed with target complementation, configuration transformation, enzyme recognition, and signal reporting abilities. The presence of a specific target, the model of miRNA-21, to react with MF-HP would cause an intermolecular priming-directed strand replication in parallel with an intramolecular priming-directed strand replication. The co-directed priming pattern results in the occurrence of SM-SDA containing a target-induced cyclical strand displacement amplification (T-CSDA), a target analogue primer (TAP)-induced CSDA (TAP-CSDA), and a nicked trigger primer (NTP)-induced CSDA (NTP-CSDA). The resulting multiple circuits with a synergetic signal amplification capacity remarkably enhance the target miRNA response, which has not only improved the sensitivity for qualitative and quantitative detection of miRNA but also realized the analysis of target miRNA from real clinical samples. As a proof-of-concept study, this simple but attractive isothermal signal amplification system holds a great potential for molecular diagnosis of diseases and will stimulate interest, new ideas, and discoveries in this fascinating field.

One-Step Nucleic Acid Amplification System in Comparison to the Intraoperative Frozen Section and Definitive Histological Examination Among Breast Cancer Patients: A Retrospective Survival Study.

Simple SummaryImplementing intraoperative assessment of sentinel lymph nodes by one-step nucleic acid amplification in early breast cancer can reduce the surgical burden to the patient and the costs to the health system. However, only limited data are available in terms of long-term disease-free survival and overall survival. Therefore, this study aims to compare disease-free survival and overall survival between one-step nucleic acid amplification, frozen section, and definitive histology. These results could impact the healthcare community, adding further proof to the body of evidence supporting the broader adoption of this innovative technology that enables a safe reduction in patient surgical burden and healthcare costs.BackgroundThe one-step nucleic acid amplification (OSNA) system is a novel molecular technique, which consents to quick intraoperative detection of sentinel lymph node metastases by the amplification of cytokeratin 19 mRNA. Our study aims to evaluate the OSNA method in comparison with frozen section (FS) and definitive histological examination of the sentinel lymph node biopsy among early breast cancer patients considering disease-free survival (DFS) and overall survival (OS).MethodsIn this study, we included all women who underwent sentinel lymph node biopsy (SLNB) for breast cancers classified as TNM stage I and II in our center between January 2005 and January 2017, and the follow-up was collected up to January 2019. We divided patients among three groups based on SLNB evaluation: definitive histological examination, intra-operative FS, or OSNA.ResultsWe included 2412 SLNBs: 727 by definitive histological examination, 697 by FS, and 988 by OSNA. Isolated tumor cells were found in 2.32% of cases, micrometastasis in 9.12%, and macrometastases in 13.64%. Surgical procedure duration was significantly shorter in OSNA than in FS (42.1 minutes ±5.1 vs. 70.1 minutes ±10.5, p <0.05). No significant differences have been observed among the three groups regarding OS, DSF, cumulative local, or distant metastases. In particular 5-year DFS was 96.38% in definitive histology (95% C.I. 95.02-97.75%), 96.37% in FS (95% C.I. 94.98-97.78%), and 96.51% in OSNA group (95% C.I. 95.32-97.72%).ConclusionsNo difference in OS and DFS was found comparing OSNA, FS, and definitive histology. Furthermore, reduced operative time was found in the OSNA group.

Ligation-free isothermal nucleic acid amplification

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Detection of lymph node metastasis in non-small cell lung cancer using the new system of one-step nucleic acid amplification assay.

IntroductionThe prognosis of non-small cell lung cancer greatly depends on the presence of lymph node metastasis, which limits the need for surgery and adjuvant therapy for advanced cancer. One-step nucleic acid amplification of cytokeratin19 (CK19) mRNA was used to detect lymph node metastasis. Automated Gene Amplification Detector RD-200 and the LYNOAMP CK19 gene amplification reagent as components of the new one-step nucleic acid amplification system, which has increased gene amplification efficiency by improving the reagent composition, have shorter preprocessing and measurement times than conventional systems. We aimed to compare the clinical performance of the new system with that of histopathology and the conventional system.Materials and methods199 lymph nodes from 58 non-small cell lung cancer patients who underwent lymph node dissection were examined intraoperatively using the new system, conventional system, and histopathology.ResultsLymph node metastasis was diagnosed in 32, 42, and 44 patients using histopathological analysis, the new system, and the conventional system, respectively. Compared with histopathological analysis, the concordance rate, sensitivity, specificity, positive predictive value, and negative predictive value of the new system were 92.0%, 90.6%, 92.2%, 69.0%, and 98.1%, respectively, and compared with the conventional system, the values were 95.0%, 86.4%, 97.4%, 90.5%, and 96.2%, respectively.ConclusionThe clinical performance of the new one-step nucleic acid amplification system in detecting lymph node metastasis of lung cancer is comparable to that of histopathology and the conventional system; its performance was sufficient for determining the appropriate clinical treatment. The new rapid system can be effectively utilized during lung cancer treatment intraoperatively and postoperatively.

Verification of lateral flow antigen tests for SARS-CoV-2 by qPCR directly from the test device

ObjectiveFast and reliable detection of infection is a key to control the SARS-CoV-2 pandemic. Lateral flow antigen tests (LFATs) are inexpensive, easy to use, but have to be verified, as they are rather unspecific and can produce both, false positive and false negative results. Our objective was to combine the speed of LFAT for SARS-CoV-2 with the reliability of qPCR tests. MethodsA serial dilution of a patient sample positive for SARS-CoV-2 was prepared and added to LFAT wells from two manufacturers. After evaluation, the devices were opened, the strips removed and extracted in a solution. Amplification was performed using point of care PCR systems (cobas® Liat®, ID NOW™) or on a LightCycler after extraction by MagNAPure 96. ResultsThe nucleic acid amplification systems yielded higher sensitivity to LFAT. Thus, all samples determined positive by LFAT from the serial dilution were also positive in the subsequent amplification reactions. Sensitivity using extracted eluates was 10–100 times higher. SignificanceThe usage of LFAT is highly recommended for single samples in emergency dental or emergency clinical settings, for smaller cohorts, or even for larger population screening, as it is inexpensive and fast. Positive results can be conveniently verified directly from the test devices using either point of care test equipment or more complex laboratory equipment thus making a major impact on efficient management of infections and isolations.

Overview of the Clinical and Molecular Features of Legionella Pneumophila: Focus on Novel Surveillance and Diagnostic Strategies.

Legionella pneumophila (L. pneumophila) is one of the most threatening nosocomial pathogens. The implementation of novel and more effective surveillance and diagnostic strategies is mandatory to prevent the occurrence of legionellosis outbreaks in hospital environments. On these bases, the present review is aimed to describe the main clinical and molecular features of L. pneumophila focusing attention on the latest findings on drug resistance mechanisms. In addition, a detailed description of the current guidelines for the disinfection and surveillance of the water systems is also provided. Finally, the diagnostic strategies available for the detection of Legionella spp. were critically reviewed, paying the attention to the description of the culture, serological and molecular methods as well as on the novel high-sensitive nucleic acid amplification systems, such as droplet digital PCR.

Development and validation of LAMP primer sets for rapid identification of Aspergillus fumigatus carrying the cyp51A TR46 azole resistance gene

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. The principal class of azole-resistant isolates is characterized by tandem repeats of 34 bp or 46 bp within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid amplification system that is fast and specific. Here we describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets. It also differentiated strains with TR46 tandem repeats from those with TR34 tandem repeats. These results showed this TR46-LAMP method is specific, rapid, and provides crucial insights to develop novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR46 tandem repeats.

Development of amplification system for point-of-care test of nucleic acid

Nucleic acid testing (NAT) has been widely used in many fields such as medical diagnosis, food safety testing and forensic identification. However, it can only be carried out in professional laboratory because the test process is complicated and rigorous. In this paper, a nucleic acid amplification system based on polymerase chain reaction (PCR) was developed to meet the requirements of point-of-care testing (POCT) for nucleic acids. Firstly, the mechanical structure and electronic control system were designed and constructed. Secondly, an integral separation PID algorithm for temperature control and an intelligent temperature compensation method based on support vector regression (SVR) were proposed. Finally, temperature measurement and biological experiments were performed to prove the stability and availability of the nucleic acid amplification system. The results showed that the system achieved a rapid temperature change velocity of 4.5 °C/s, and the steady-state error was within ± 0.5 °C. The nucleic acids in samples of different concentrations were well amplified, the system can be used for quantitative detection of nucleic acid with the help of a fluorescence detection system, and has higher sensitivity than Tianlong PCR instrument.

A novel nucleic acid amplification system based on nano-gap embedded active disk resonators

Recent advances in nucleic acid based testing using bio-optical sensor approaches have been introduced but most are based on hybridization between the optical sensor and the bio-molecule and not on an amplification mechanism. Direct nucleic acid amplification on an optical sensor has several technical limitations, such as the sensitivity of the temperature sensor, instrument complexity, and high background signal. We here describe a novel nucleic acid amplification method based on a whispering gallery mode active resonator and discuss its potential molecular diagnostic application. By implanting nanoclusters as active compounds, this active resonator operates without tapered fiber coupling and emits a strong photoluminescence signal with low background in the wavelength of low absorption in an aqueous environment that is typical of biosensors. Our method also offers an extremely low detection threshold down to a single copy within 10 min due to the strong light-matter interaction in a nano-gap structure. We envision that this active resonator provides a high refractive index contrast for tight mode confinement with simple alignment as well as the possibility of reducing the device size so that a point-of-care system with low-cost, high-sensitivity and simplicity.

Microfluidic-CFPA Chip for the Point-of-Care Detection of African Swine Fever Virus with a Median Time to Threshold in about 10 min.

The outbreak of African swine fever has brought serious public safety problems to the world, and there is a need for reliable diagnostic tools to prevent and control the epidemic. Here, we successfully established a portable microfluidic-circular fluorescent probe-mediated isothermal nucleic acid amplification (CFPA) system for the rapid, high-throughput, accurate, and sensitive detection of African swine fever virus at point-of-care settings. This detection system has a detection limit of 10 copies/μL, good stability (C.V. < 5%), and 92.73% sensitivity, and 100% specificity when tested on 220 pig samples collected for the diagnosis of African swine fever virus, with a median time to threshold of 10.8 min. This novel integrated diagnostic tool is urgently needed and has promising applications for the monitoring and control of African swine fever epidemics worldwide and, in particular, the serious outbreak in China.

Microfluidic-Based Nucleic Acid Amplification Systems in Microbiology.

Rapid, sensitive, and selective bacterial detection is a hot topic, because the progress in this research area has had a broad range of applications. Novel and innovative strategies for detection and identification of bacterial nucleic acids are important for practical applications. Microfluidics is an emerging technology that only requires small amounts of liquid samples. Microfluidic devices allow for rapid advances in microbiology, enabling access to methods of amplifying nucleic acid molecules and overcoming difficulties faced by conventional. In this review, we summarize the recent progress in microfluidics-based polymerase chain reaction devices for the detection of nucleic acid biomarkers. The paper also discusses the recent development of isothermal nucleic acid amplification and droplet-based microfluidics devices. We discuss recent microfluidic techniques for sample preparation prior to the amplification process.