Nucleic Acid Detection Techniques Research Articles

A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick

Tick-borne encephalitis virus (TBEV), a zoonotic pathogen, can cause severe neurological complications and fatal outcomes in humans. Early diagnosis of TBEV infection is crucial for clinical practice. Although serological assays are frequently employed for detection, the lack of antibodies in the early stages of infection and the cross-reactivity of antibodies limit their efficacy. Conventional molecular diagnostic methods such as RT-qPCR can achieve early and accurate identification but require specialized instrumentation and professionals, hindering their application in resource-limited areas. Our study developed a rapid and visual TBEV molecular detection method by combining RT-recombinase-aided amplification, the CRISPR/Cas13a system, and lateral flow dipsticks. The diagnostic sensitivity of this method is 50 CFU/ml, with no cross-reactivity with a variety of viruses. The detection can be carried out within 1 h at a temperature between 37 and 42 °C, and the results can be visually determined without the need for complex instruments and professionals. Subsequently, this assay was used to analyze clinical samples from 15 patients suspected of TBEV infection and 10 healthy volunteers, and its sensitivity and specificity reached 100 %, which was consistent with the results of RT-qPCR. These results indicate that this new method can be a promising point-of-care test for the diagnosis of tick-borne encephalitis.

Study on the applicability of novel molecular targets of Vibrio species by pan-genome mining in the field of nucleic acid detection

Vibrio spp. are ubiquitous in marine areas worldwide and can cause diseases in humans. Aqueous environments favor the transfer of genes between strains, resulting in the loss of strain-specific target genes. This study aimed to explore new molecular targets for the detection of Vibrio strains at the species level using pan-genome analysis for nucleic acid detection and differentiation of major pathogenic Vibrio in aquatic products. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: three for Vibrio parahaemolyticus, one for Vibrio alginolyticus, three for Vibrio vulnificus, three for Vibrio fluvialis. We constructed primers for the selected genes, and we verified their specificity using various Vibrio species and non-Vibrio strains. After systematic optimization, a novel molecular target-based 4-multiplex PCR (4-mPCR) method was developed, exhibiting a detection limit of 103 CFU/mL for pure cultures and 104 CFU/g for fish samples. The initial inoculum was approximately 1 Log10 CFU/g, and all samples were positive after 4 h of enrichment. Not only that, the feasibility of the mined new targets in the field of nucleic acid detection was further validated by the 4-mqPCR and LAMP techniques. These results suggest that novel targets mined by pan-genomic have good generalizability for use in a variety of nucleic acid detection techniques and can help improve the accuracy of nucleic acid-based detection techniques in the detection of pathogenic Vibrio species in aquatic samples, with potential applications for food safety investigations and disease diagnosis.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Photochromic visual sensing chip for isothermal amplification detection of porcine transmissible gastroenteritis virus

The outbreak of transmissible gastroenteritis virus (TGEV) will cause huge economic losses to the whole pig industry. Hence, there is urgent need to develop a rapid and ultrasensitive method for detection of TGEV. As a nucleic acid detection technique, loop-mediated isothermal amplification (LAMP) can achieve quantitative detection of targeted nucleic acids with high sensitivity and selectivity. Nevertheless, the signal outputs of LAMP method must be acquired by complicated instruments. In this work, we firstly developed a LAMP photochromic sensing chip for porcine TGEV detection by combination of the photochromic sensing chip and nucleic acid amplification. The detection signal was based on color change of electrochromic material rather than electrical signal, and thus the detection signal can be obtained by visualization without relying on complicated instrument. The entire test was performed with small fluorinated indium tin oxide electrodes modified with zinc oxide (ZnO) (a photocatalytic material) and Prussian blue (PB) (an electrochromic material). When photoinduced electrons produced by ZnO were injected into PB under light, the PB was reduced to Prussian white. The higher the concentration of TGEV, the more double-stranded DNA was produced after amplification. The amplified product produced greater impedance, and fewer electron was transferred, which affect the corresponding color change of PB. The sensing chip also showed highly sensitive response to TGEV, with the minimum limit of detection was determined to be 2.5 fg/μL. The sensing chip developed herein will provide a new avenue for DNA amplification detection by visualization.

CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

AbstractPolymerase chain reaction (PCR)‐based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR‐mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point‐of‐care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR‐based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics‐target preamplification‐free detection, microRNA (miRNA) testing, non‐nucleic‐acid detection, and SARS‐CoV‐2 detection‐are also covered.

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) testing, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.

A rapid nucleic acid detection platform based on phosphorothioate-DNA and sulfur binding domain

Nucleic acid detection plays a key role in diverse diagnosis and disease control. Currently available nucleic acid detection techniques are challenged by trade-offs among speed, simplicity, precision and cost. Here, we described a novel method, designated SENSOR (Sulfur DNA mediated nucleic acid sensing platform), for rapid nucleic acid detection. SENSOR was developed from phosphorothioate (PT)-DNA and sulfur binding domain (SBD) which specifically binds double-stranded PT-modified DNA. SENSOR utilizes PT-DNA oligo and SBD as targeting module, which is linked with split luciferase reporter to generate luminescence signal within 10 min. We tested detection on synthesized nucleic acid and COVID-19 pseudovirus, achieving attomolar sensitivity combined with an amplification procedure. Single nucleotide polymorphisms (SNP) could also be discriminated. Indicating SENSOR a new promising nucleic acid detection technique.

Rolling Circle Amplification on a Bead: Improving the Detection Time for a Magnetic Bioassay.

Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometer-sized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.

MicroRNA Detection with CRISPR/Cas.

Low-cost detection of miRNAs has caught broad attention in recent years due to the potential application of these small noncoding RNAs for diagnostics and therapeutic purposes. Their small size and low abundance, however, derive challenges in engineering robust detection tools. To date, multiple detection assays have been developed to achieve highly specific recognition of trace amount of miRNA with state-of-the-art nucleic acid detection and signal amplification techniques. In this chapter we describe how isothermal amplification techniques and CRISPR/Cas-based techniques can be integrated to generate rationally designed miRNA detection systems for specific miRNA.

Tools to Alleviate the Drug Resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), an acid-fast bacillus that causes Tuberculosis (TB), is a pathogen that caused 1.5 million deaths in 2020. As per WHO estimates, another 4.1 million people are suffering from latent TB, either asymptomatic or not diagnosed, and the frequency of drug resistance is increasing due to intrinsically linked factors from both host and bacterium. For instance, poor access to TB diagnosis and reduced treatment in the era of the COVID-19 pandemic has resulted in more TB deaths and an 18% reduction in newly diagnosed cases of TB. Additionally, the detection of Mtb isolates exhibiting resistance to multiple drugs (MDR, XDR, and TDR) has complicated the scenario in the pathogen’s favour. Moreover, the conventional methods to detect drug resistance may miss mutations, making it challenging to decide on the treatment regimen. However, owing to collaborative initiatives, the last two decades have witnessed several advancements in both the detection methods and drug discovery against drug-resistant isolates. The majority of them belong to nucleic acid detection techniques. In this review, we highlight and summarize the molecular mechanism underlying drug resistance in Mtb, the recent advancements in resistance detection methods, and the newer drugs used against drug-resistant TB.

Protocolo diagnóstico y tratamiento de la neumonía en el paciente inmunocomprometido

Advances in oncological treatment, immunosuppressive therapies for preventing graft versus host disease, and the widespread use of therapies with biological or immunomodulating drugs has led to an increase in the immunocompromised population in recent years. Severe respiratory infections are the main cause of ICU admission in immunocompromised patients. In regard to the diagnostic approach, the time since onset of the lung disease, the extension of immunosuppression, and radiological patterns will be considered. The development of nucleic acid detection techniques and their application in these patients improves diagnostic sensitivity. It is very important to start empirical treatment as soon as possible in these patients, provided that it is possible after collecting samples.

Single-Molecule Detection of Nucleic Acids via Liposome Signal Amplification in Mass Spectrometry.

A single-molecule detection method was developed for nucleic acids based on mass spectrometry counting single liposome particles. Before the appearance of symptoms, a negligible amount of nucleic acids and biomarkers for the clinical diagnosis of the disease were already present. However, it is difficult to detect extremely low concentrations of nucleic acids using the current methods. Hence, the establishment of an ultra-sensitive nucleic acid detection technique is urgently needed. Herein, magnetic beads were used to capture target nucleic acids, and liposome particles were employed as mass tags for single-particle measurements. Liposomes were released from magnetic beads via photocatalytic cleavage. Hence, one DNA molecule corresponded to one liposome particle, which could be counted using mass spectrometric measurement. The ultrasensitive detection of DNA (10–18 M) was achieved using this method.

Current Trends of SARS-CoV-2 and its New Variants Diagnostics in Different Body Fluids: Surface Antigen, Antibody, Nucleic Acid, and RNA Sequencing Detection Techniques

Current Trends of SARS-CoV-2 and its New Variants Diagnostics in Different Body Fluids: Surface Antigen, Antibody, Nucleic Acid, and RNA Sequencing Detection Techniques

Development of serological assay for detection of antibodies in the N protein of SARS-CoV-2 in human sera in Mongolia

The capability to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and identify immune responses among the population is crucial for managing the outbreak of the COVID-19 pandemic. Although PCR-based nucleic acid detection techniques are utilized to detect viral infection in people, alternative tests capable of distinguishing between exposure and infection are urgently needed beyond this restricted window of detectable viral replication. Antibodies are produced in human sera within a few days after viral infection, providing longer period for performing tests to acquire reliable database. Herewith, we provide the results of our in-house developed ELISA (Enzyme-Linked Immunosorbent Assay) that displays all of the properties necessary for high-throughput of human sera sample analysis. This test does not involve the handling of live viruses, although it detects a variety of antibody types in serum and plasma of human after exposure to the virus. For in-house development of the kit, the nucleocapsid (N) gene of SARS-CoV-2 virus was cloned in the prokaryotic expression vector pGEX-6P-1, and purified N protein was used to detect IgG antibodies in human sera samples. In total 76 human serum samples that were collected before novel coronavirus registry in Mongolia in March 2020, as well as 200 serum samples from patients who had been infected by SARS-CoV-2 virus, were used. Among 200 serum samples, 188 were positive and 12 were false negative, while in non-infected cases 69 were negative and 7 were false positive, suggesting 94 per cent sensitivity and 90.7 per cent specificity of the kit, with p-values of 0.02.

Methodological study on eliminating nucleic acid contamination in molecular diagnostic laboratory

Nucleic acid detection technique has good sensitivity and specificity and is widely used in in vitro diagnosis, animal and plant commodity quarantine, forensic identification, and other fields. However, it is susceptible to carryover contamination during the operation and leads to false-positive results, which seriously affects the detection accuracy. Therefore, finding an effective solution to prevent and eliminate nucleic acid carryover contamination has become particularly urgent. This study compared several different methods for removing nucleic acid contamination and confirmed that sodium hypochlorite solution and PCRguard reagent could effectively eliminate nucleic acid carryover in the liquid and on surfaces of different materials. Besides, the combination of sodium hypochlorite solution and PCRguard can solve the nucleic acid aerosol contamination. This study proposes solutions for the routine prevention of carryover contamination and removal of aerosol that has occurred in molecular diagnostic laboratories.

CRISPR-Assisted DNA Detection: A Novel dCas9-Based DNA Detection Technique.

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.

Turfgrass Disease Diagnosis: Past, Present, and Future.

Turfgrass is a multibillion-dollar industry severely affected by plant pathogens including fungi, bacteria, viruses, and nematodes. Many of the diseases in turfgrass have similar signs and symptoms, making it difficult to diagnose the specific problem pathogen. Incorrect diagnosis leads to the delay of treatment and excessive use of chemicals. To effectively control these diseases, it is important to have rapid and accurate detection systems in the early stages of infection that harbor relatively low pathogen populations. There are many methods for diagnosing pathogens on turfgrass. Traditional methods include symptoms, morphology, and microscopy identification. These have been followed by nucleic acid detection and onsite detection techniques. Many of these methods allow for rapid diagnosis, some even within the field without much expertise. There are several methods that have great potential, such as high-throughput sequencing and remote sensing. Utilization of these techniques for disease diagnosis allows for faster and accurate disease diagnosis and a reduction in damage and cost of control. Understanding of each of these techniques can allow researchers to select which method is best suited for their pathogen of interest. The objective of this article is to provide an overview of the turfgrass diagnostics efforts used and highlight prospects for disease detection.

Research Progress of Nucleic Acid Detection Technology Platforms for New Coronavirus SARS-CoV-2

The new coronavirus SARS-CoV-2 has spread to the whole world, seriously threatening human life and disrupting people's lives. SARS-CoV-2 is a highly infectious virus with a long incubation period, encompassing asymptomatic infections. Therefore, accurate detection of SARS-CoV-2 is essential for the prevention and control of the epidemic. Currently, nucleic acid detection has played an important role in the prevention and control of SARS-CoV-2. A variety of nucleic acid detection technologies for SARS-CoV-2 have been developed, and some technologies have been converted into available kits for clinical detection. However, these technologies have different principles and different detection platforms. How to choose the appropriate nucleic acid detection technology for SARS-CoV-2 perplexes epidemic prevention and control. Based on the latest research progresses of nucleic acid technologies for SARS-CoV-2, this paper introduced the principles and technology platforms of these detection technologies, thoroughly compared the advantages and disadvantages of each technique and clarified the scope of applications, providing a reference for selecting the appropriate nucleic acid detection technology for SARS-CoV-2. Furthermore, this paper provided a prospect for developing technologies of detecting pathogens similar to SARS-CoV-2.

Advances in Viral Diagnostic Technologies for Combating COVID-19 and Future Pandemics.

The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens the health of the global population and challenges our preparedness for pandemic threats. Previous outbreaks of coronaviruses and other viruses have suggested the importance of diagnostic technologies in fighting viral outbreaks. Nucleic acid detection techniques are the gold standard for detecting SARS-CoV-2. Viral antigen tests and serological tests that detect host antibodies have also been developed for studying the epidemiology of COVID-19 and estimating the population that may have immunity to SARS-CoV-2. Nevertheless, the availability, cost, and performance of existing viral diagnostic technologies limit their practicality, and novel approaches are required for improving our readiness for global pandemics. Here, we review the principles and limitations of major viral diagnostic technologies and highlight recent advances of molecular assays for COVID-19. In addition, we discuss emerging technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR) systems, high-throughput sequencing, and single-cell and single-molecule analysis, for improving our ability to understand, trace, and contain viral outbreaks. The prospects of viral diagnostic technologies for combating future pandemic threats are presented.

Preliminary recommendations for lung surgery during COVID-19 epidemic period.

Preliminary recommendations for lung surgery during COVID-19 epidemic period.