Rapid Nucleic Acid Research Articles

Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing

Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens (n = 121).

Clinical feature of omicron infection in children with inborn errors of immunity in China.

SARS-CoV-2 infection is hypothesized to be more severe in immunocompromised patients; however, clinical outcomes in children with inborn errors of immunity (IEI) during the Omicron pandemic in China have not been reported. This cohort study retrospectively reviewed 71 SARS-CoV-2-infected children with IEI using nationwide data from the National Center for Children's Health of China. COVID-19 was diagnosed by a positive rapid antigen or nucleic acid test result. Among 71 SARS-CoV-2-infected children with IEI, male preponderance (male: female ratio of ~1.8:1), a median age of 8 years (IQR 3-11), and a predominance of antibody deficiency (19/71, 26.8%) were detected. Most of the patients got infected through household transmission, while a small proportion of them did so during hospital visits. The mean time periods were 3.3 days (n=44) for incubation, 8.4 days for symptoms (n=69), and 8.8 days for viral shedding (n=37). The time to viral shedding was proportional to the symptomatic period (R2 = 0.1243, p=0.0323) and prolonged in children with X- linked agammaglobulinemia. The most common symptoms of COVID-19 were fever, and some children showed only aggravation of the underlying disease. 15% of IEI children progress to pneumonia, 85% require medication, 17% are admitted to hospital, and 4.1% are classified as critical. Previously application of anti- infective medications was associated with an increased risk of hospitalization after COVID-19 infection. Of the 71 children with IEI, all recovered from COVID- 19. Overall, Omicron variant did not cause significant life-threatening infections among children with IEI in China, and most of them had a good clinical outcome. Nevertheless, these children exhibit an increased vulnerability to higher hospitalization rates, pneumonia, and severe illness compared to the general pediatric population.

A one-pot loop-mediated isothermal amplification platform using fluorescent gold nanoclusters for rapid and naked-eye pathogen detection

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive nucleic acid testing method for pathogen detection, yet the absence of a straightforward readout strategy remains challenging. We've successfully designed polyethyleneimine-stabilized gold nanoclusters (PEI-AuNCs) as a cationic AuNCs indicator tailored for distinguishing LAMP results, enabling direct visual inspection under UV light. Positive LAMP reactions with PEI-AuNCs, in combination with magnesium pyrophosphate crystals, yield red-fluorescent bulk precipitates visible to the naked eye. To address contamination concerns, we introduced a one-pot reaction by incorporating AuNCs into the lid recess. This one-pot LAMP assay demonstrates exceptional detection capability, identifying Salmonella enterica at concentrations as low as 101 CFU/mL within approximately 50 min, excluding nucleic acid extraction. The platform's versatility, achieved through customizable primers, positions it as a promising molecular diagnostic tool for rapid and visual pathogen detection across scientific disciplines.

Development of an Integrated Disposable Device for SARSCoV-2 Nucleic Acid Extraction and Detection

ObjectiveTo develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MethodsWe designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. ResultsThe precision of the syringe transfer volume was 19.2 ± 1.9 μL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov- 2 nucleic acid extraction yield through the device was 7.10 × 106 copies/mL, while a commercial kit yielded 2.98 × 106 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. ConclusionThe integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Advancements and applications of loop-mediated isothermal amplification technology: a comprehensive overview.

Loop-mediated isothermal amplification (LAMP) is a novel method for nucleic acid detection known for its isothermal properties, high efficiency, sensitivity, and specificity. LAMP employs 4 to 6 primers targeting 6 to 8 regions of the desired sequence, allowing for amplification at temperatures between 60 and 65°C and the production of up to 109 copies within a single hour. The product can be monitored by various methods such as turbidimetry, fluorometry, and colorimetry. However, it faces limitations such as the risk of non-specific amplification, challenges in primer design, unsuitability for short gene sequences, and difficulty in multiplexing. Recent advancements in polymerase and primer design have enhanced the speed and convenience of the LAMP reaction. Additionally, integrating LAMP with technologies like rolling circle amplification (RCA), recombinase polymerase amplification (RPA), and CRISPR-Cas systems has enhanced its efficiency. The combination of LAMP with various biosensors has enabled real-time analysis, broadening its application in point-of-care testing (POCT). Microfluidic technology has further facilitated the automation and miniaturization of LAMP assays, allowing for the simultaneous detection of multiple targets and preventing contamination. This review highlights advancements in LAMP, focusing on primer design, polymerase engineering, and its integration with other technologies. Continuous improvements and integration of LAMP with complementary technologies have significantly enhanced its diagnostic capabilities, making it a robust tool for rapid, sensitive, and specific nucleic acid detection with promising implications for healthcare, agriculture, and environmental monitoring.

Simple and rapid identification of beef within 30 min using a new food nucleic acid release agent combined with direct-fast qPCR

Simple and rapid molecular detection technologies for authenticating animal species are urgently needed for food safety and authenticity. This study established a new direct-fast quantitative polymerase chain reaction (qPCR) detection technology for beef to achieve rapid and on-site nucleic acid detection in food. This technology can complete nucleic acid extraction in 4 min using a new type of food nucleic acid-releasing agent, followed by direct amplification of the DNA sample by fast qPCR in 25 min. The results indicated that direct-fast qPCR can specifically identify beef and can also identify 0.00001% of beef components in artificially simulated meat mixtures, with a detection precision variation coefficient of <4%. This method can be used to effectively identify beef in different food samples. As a simple, fast, and accurate molecular detection technology for beef, this method may provide a new tool for the on-site detection of beef components in food.

Minute level ultra-rapid and thousand copies level high-sensitive pathogen nucleic acid identification based on contactless impedance detection microsensor

Early screening for pathogens is crucial during pandemic outbreaks. Nucleic acid testing (NAT) is a valuable method for keeping pathogens from spreading. However, the long detection time and large size of the instruments involved significantly limited the efficiency of detection. This work described an integrated NAT microsensor that facilitated rapid and extremely sensitive detection based on nucleic acid amplification (NAA) on a chip. The biochip consisted of two layers incorporating a heater, a thermometer, an interdigital electrode (IDE) and a reaction chamber. The Pt electrode based heater and thermometer were utilized to maintain a specific temperature for the sample in the chamber. The thermometer exhibited a good linear correlation with a sensitivity of 9.36 Ω/°C and the heater achieved a heating efficiency of approximately 6.5 °C/s. Multiple ions were released during NAA, resulting in a decrease in the impedance of the amplification system solution. A large signal of impedance was generated by the released ions due to its linear correlation with the logarithm of the ion concentration. With this detection principle, IDE was employed for real-time monitoring of the in-chip reaction system impedance and NAA process. Specific nucleic acids from two pathogens (SARS-CoV-2, Vibrio vulnificus) were detected with this microsensor. The samples were qualitatively analyzed on microchip within 3 min, with a limit of detection (LOD) of 103 copies/μL. The proposed sensor presented several advantages, including reduced NAT time and increased sensitivity. Consequently, it has shown significant potential in rapid and high-quality nucleic acid testing for the field of epidemic prevention.

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.

Rapid and Amplification-free Nucleic Acid Detection with DNA Substrate-Mediated Autocatalysis of CRISPR/Cas12a.

To enable rapid and accurate point-of-care DNA detection, we have developed a single-step, amplification-free nucleic acid detection platform, a DNA substrate-mediated autocatalysis of CRISPR/Cas12a (DSAC). DSAC makes use of the trans-cleavage activity of Cas12a and target template-activated DNA substrate for dual signal amplifications. DSAC employs two distinct DNA substrate types: one that enhances signal amplification and the other that negatively modulates fluorescent signals. The positive inducer utilizes nicked- or loop-based DNA substrates to activate CRISPR/Cas12a, initiating trans-cleavage activity in a positive feedback loop, ultimately amplifying the fluorescent signals. The negative modulator, which involves competitor-based DNA substrates, competes with the probes for trans-cleaving, resulting in a signal decline in the presence of target DNA. These DNA substrate-based DSAC systems were adapted to fluorescence-based and paper-based lateral flow strip detection platforms. Our DSAC system accurately detected African swine fever virus (ASFV) in swine's blood samples at femtomolar sensitivity within 20 min. In contrast to the existing amplification-free CRISPR/Dx platforms, DSAC offers a cost-effective and straightforward detection method, requiring only the addition of a rationally designed DNA oligonucleotide. Notably, a common ASFV sequence-encoded DNA substrate can be directly applied to detect human nucleic acids through a dual crRNA targeting system. Consequently, our single-step DSAC system presents an alternative point-of-care diagnostic tool for the sensitive, accurate, and timely diagnosis of viral infections with potential applicability to human disease detection.

Rapid fluorescent nucleic acid sensing with ultra-thin gold nanosheets

Fluorescently labeled DNA oligonucleotides and gold nanospheres have been frequently utilized in biosensors, providing efficient nucleic acid detection. Nevertheless, the restricted loading capacity of gold nanospheres undermines overall sensitivity. In this study, we employed four-atom-thick ultrathin gold nanosheets (AuNSs), utilizing a “pre-mix model” for rapid target nucleic acid detection. In this approach, fluorescently labeled DNA probes were pre-incubated with the target nucleic acid, followed by the addition of AuNSs for probe adsorption and fluorescence quenching. With the developed method, we efficiently and rapidly detected the SARS-CoV-2 N gene sequence within 30 min, involving a brief 15-min target pre-incubation and a subsequent 15-min adsorption of free probes and fluorescence quenching by AuNSs. This method exhibited heightened sensitivity compared to gold nanospheres, boasting a limit of detection (LOD) of 0.808 nM. Furthermore, exceptional recovery was achieved in simulated biological samples. The study introduces an effective strategy for nucleic acid sensing characterized by rapidity, heightened sensitivity, ease of operation, and robustness. These findings encourage further development of rapid biomarker sensing methods employing 2D nanomaterials.

Rapid and accurate detection of fish allergen within 30 min using a food nucleic acid releaser combined with rapid real time PCR assay

To achieve rapid on-site nucleic acid detection of fish allergens in food, this study established a new direct and rapid qPCR detection technology for fish. The technology was able to complete nucleic acid extraction in 4 min using a food nucleic acid releaser, and the DNA samples were amplified by rapid qPCR within 25 min. The results showed that direct-rapid qPCR can specifically detect fish, and it can also detect 0.00001 % of fish components in artificially simulated samples, with high detection precision. This method can be used to effectively detect fish in various deep-processed foods. As a simple, rapid, and accurate molecular detection method for fish allergens, this method has broad application prospects for on-site food safety detection.

SYPHILIS IN THE 21ST CENTURY: CHALLENGES AND ADVANCES IN DIAGNOSIS AND TREATMENT

The article titled Syphilis in the 21st Century: Challenges and Advances in Diagnosis and Treatment aimed to analyze the main challenges and significant advances in the diagnosis and treatment of syphilis throughout the 21st century. The research focused on understanding how new technologies and public health strategies have contributed to the improvement of disease management, as well as identifying the existing barriers. A bibliographic review was conducted to compile and synthesize relevant data from previous studies, providing a comprehensive overview of the topic. The explanatory methodology used allowed for a theoretical deepening of the issues involved in the detection and treatment of syphilis. The technical procedures included the analysis of scientific articles, health organization reports, and systematic reviews that addressed both technological advances and the challenges faced by health professionals. Among the main advances discussed were rapid tests and nucleic acid amplification tests (NAATs), which have significantly improved the ability to diagnose the infection early and accurately. Improvements in public awareness and prevention programs were also addressed. Despite the advances, the article also highlighted persistent challenges such as antibiotic resistance and difficulties in accessing effective treatments, especially in less developed regions. Benzathine penicillin G was reaffirmed as the treatment of choice, but distribution logistics and adherence to treatment remain important obstacles. The bibliographic review concluded that, for effective control of syphilis, integrated strategies that combine education, prevention, early diagnosis, and adequate treatment are necessary, as indicated in the reviewed literature.

RNA extraction-free reduced graphene oxide-based RT-LAMP fluorescence assay for highly sensitive SARS-CoV-2 detection

Infectious diseases have always been a seriously endanger for human life and health. A rapid, accurate and ultra-sensitive virus nucleic acid detection is still a challenge to deal with infectious diseases. Here, a RNA extraction-free reduced graphene oxide-based reverse transcription-loop-mediated isothermal amplification (EF-G-RT-LAMP) fluorescence assay was developed to achieve high-throughput, rapid and ultra-sensitive SARS-CoV-2 RNA detection. The whole detection process only took ∼36 min. The EF-G-RT-LAMP assay achieves a detection limit of 0.6 copies μL−1 with a wide dynamic range of aM-pM. A large number (up to 384) of samples can be detected simultaneously. Simulated detection of the COVID-19 pseudovirus and clinical samples in nasopharyngeal swabs demonstrated a high-throughput, rapid and ultra-sensitive practical detection capability of the EF-G-RT-LAMP assay. The results proved that the assay would be used as a rapid, easy-to-implement approach for epidemiologic diagnosis and could be extended to other nucleic acid detections.

Evaluation of Abbott ID-NOW Rapid Assay with Real-Time Reverse Transcription PCR Assay for Detection of SARS-CoV-2

Rapid nucleic acid assays have been approved by FDA for managing the COVID-19 pandemic, however its analytical efficiency has not been thoroughly validated. This study evaluates the detection and identification of COVID-19 virus using Abbott ID-Now to rapidly identify cases and intervention practices in comparison to nucleic acid detection. Nasopharyngeal Swabs collected from 611 participants were tested for Abbott ID-NOW and LabGun COVID-19 ExoFast RT-PCR Kit as per manufacturer’s protocol. The results from the ID NOW™ COVID-19 assay were evaluated by comparing results with the standard RT-PCR, which served as a standard reference. The infection burden of SARS-CoV-2 in the population of UAE was 11.62%. Compared to detection using real time-based platforms, the sensitivity, specificity, positive and negative predictive values of the ID-Now were 84.51%, 99.81%, 98.36% and 98.00% respectively for COVID-19. A stratified analysis was also carried out using cycle threshold (Ct) values categorizing as Ct&gt;33 as with low viral loads while those with Ct&lt;33 as high. This demonstrated statistically significant (P&lt;0.0001) decrease in sensitivity in samples (97.87% in low Ct value samples versus 58.33% in high Ct value samples). Even though the sensitivity for Abbott ID NOW™ in this study was lower, the specificity, positive predictive values and negative predictive values were significant in low viral load samples. It is easy to use and interpret, giving early information to support clinical decision-making ID-NOW could be possibly used as a point-of-care test after evaluation in epidemic and endemic settings.

A Portable, Integrated, Sample-In Result-Out Nucleic Acid Diagnostic Device for Rapid and Sensitive Chikungunya Virus Detection.

Chikungunya virus, a mosquito-borne virus that causes epidemics, is often misdiagnosed due to symptom similarities with other arboviruses. Here, a portable and integrated nucleic acid-based diagnostic device, which combines reverse transcription-loop-mediated isothermal amplification and lateral-flow detection, was developed. The device is simple to use, precise, equipment-free, and highly sensitive, enabling rapid chikungunya virus identification. The result can be obtained by the naked eye within 40 min. The assay can effectively distinguish chikungunya virus from dengue virus, Japanese encephalitis virus, Zika virus, and yellow fever virus with high specificity and sensitivity as low as 598.46 copies mL-1. It has many benefits for the community screening and monitoring of chikungunya virus in resource-limited areas because of its effectiveness and simplicity. The platform has great potential for the rapid nucleic acid detection of other viruses.

Water-cooling-based and low-cost qPCR device for rapid nucleic acid analysis

Real-time quantitative PCR (qPCR) is a sought-after method for quantifying nucleic acids. A series of commercial instruments based on qPCR has been developed and applied to detect several diseases. However, commercial instruments have some limitations in terms of cost, bulk, and efficiency. Herein, we proposed a novel qPCR device using water-cooling technology to shorten the period of the PCR reaction. The device was made of an amplification module with a novelty water-cooling-based PCR chip, a custom-made fluorescence detection module, and a control module based on economical off-the-shelf electronics. All these modules were assembled in a 3D-printed frame with a size of 11 cm × 9.7 cm × 8.9 cm and a weight of 0.5 kg. With the help of water-cooling technology, the total time required to complete a PCR reaction was shortened, with a maximum cooling rate of 12 ℃/s, which is much faster than the rate of conventional cooling methods. Compared with commercial qPCR instruments, our device's cost, efficiency, and portability have been significantly optimized. To test the proposed device, a qPCR experiment was successfully performed with cDNA of Vesicular stomatitis virus at concentrations ranging from 0.005 μg/mL to 50 μg/mL.

Rapid one-pot isothermal amplification reassembled of fluorescent RNA aptamer for SARS-CoV-2 detection

The outbreak of SARS-CoV-2 poses a serious threat to human life and health. A rapid nucleic acid tests can effectively curb the spread of the disease. With the advantages of fluorescent RNA aptamers, low background and high sensitivity. A variety of fluorescent RNA aptamer sensors have been developed for the detection of nucleic acid. Here, we report a hypersensitive detection platform in which SARS-CoV-2 initiates RTF-EXPAR to amplify trigger fragments. This activation leads to the reassembled of the SRB2 fluorescent RNA aptamer, restoring its secondary structure for SR-DN binding and turn-on fluorescence. The platform completes the assay in 30 min and all reactions occur in one tube. The detection limit is as low as 116 aM. Significantly, the platform's quantitative analyses were almost identical to qPCR results in simulated tests of positive samples. In conclusion, the platform is sensitive, accurate and provides a new protocol for point-of-care testing of viruses.

A novel nanoplasmonic-based diagnosis platform: Advances and emerging technologies

The development of a rapid, simple, inexpensive, and sensitive nucleic acid assay is critical in ultrafast diagnostic systems to prevent the rapid spread of novel infectious agents. However, current representative diagnostic methods, such as the polymerase chain reaction (PCR), are bulky, expensive, complex, and time-consuming. The plasmonic photothermal effect of plasmonic materials is widely used in biological applications because of excellent properties, such as strong optical properties and enhanced local electromagnetic field. PCR methods using plasmonic nanostructures are used to overcome the limitations of conventional PCR. Therefore, plasmonic photothermal-based PCR exhibit a rapid ramping rate, ultrafast amplification, high sensitivity, cost-effectiveness, and compactness, which can be used as a point-of-care device in situ. This study reviewed the mechanisms of plasmonic PCR and various materials, such as organic, inorganic, and hybrid materials, used in PCR. Furthermore, the properties, light source, amplification efficiency, and monitoring method of classified plasmonic materials were compared. This review introduced the disease diagnosis application using plasmonic PCR and evaluated the prospect of potential future applications as next-generation PCR.

METODE DIAGNOSIS INFEKSI TRICHOMONAS VAGINALIS TERKINI

Recent advances in tests for the sexually transmitted protozoan parasite Trichomonas vaginalis have increased opportunities for diagnosis and treatment of this important sexually transmitted infection. This review summarizes current available tests, highlighting their performance characteristics, advantages, and limitations. The recent development of molecular tests for the detection of T vaginalis, including rapid antigen detection and nucleic acid amplification tests, has significantly improved the quality of diagnostics for trichomoniasis, particularly in women. In light of the expanded menu of testing options now available, improved recognition and better control of trichomoniasis are in sight, which should enable the eventual reduction of adverse reproductive consequences associated with T vaginalis infection.

Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.