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1.
Biosens Bioelectron ; 219: 114816, 2022 Oct 17.
Article in English | MEDLINE | ID: covidwho-2068733

ABSTRACT

Airborne transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for aerosol monitoring of SARS-CoV-2 to prevent sporadic outbreaks of COVID-19. The inadequate sensitivity of conventional methods and the lack of an on-site detection system limited the practical SARS-CoV-2 monitoring of aerosols in public spaces. We have developed a novel SARS-CoV-2-in-aerosol monitoring system (SIAMs) which consists of multiple portable cyclone samplers for collecting aerosols from several venues and a sensitive "sample-to-answer" microsystem employing an integrated cartridge for the analysis of SARS-CoV-2 in aerosols (iCASA) near the sampling site. By seamlessly combining viral RNA extraction based on a chitosan-modified quartz filter and "in situ" tetra-primer recombinase polymerase amplification (tpRPA) into an integrated microfluidic cartridge, iCASA can provide an ultra-high sensitivity of 20 copies/mL, which is nearly one order of magnitude greater than that of the commercial kit, and a short turnaround time of 25 min. By testing various clinical samples of nasopharyngeal swabs, saliva, and exhaled breath condensates obtained from 23 COVID-19 patients, we demonstrate that the positive rate of our system was 3.3 times higher than those of the conventional method. Combining with multiple portable cyclone samplers, we detected 52.2% (12/23) of the aerosol samples, six times higher than that of the commercial kit, collected from the isolation wards of COVID-19 patients, demonstrating the excellent performance of our system for SARS-CoV-2-in-aerosol monitoring. We envision the broad application of our microsystem in aerosol monitoring for fighting the COVID-19 pandemic.

2.
BMC Vet Res ; 18(1): 369, 2022 Oct 11.
Article in English | MEDLINE | ID: covidwho-2064800

ABSTRACT

BACKGROUND: Swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute vomiting and diarrhea in piglets, leading to significant financial losses for the pig industry. Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology used under constant temperature conditions. The study established a real-time reverse transcription (RT)-RPA assay for early diagnosis of SADS-CoV.  RESULTS: The detection limit of the real-time RT-RPA was 74 copies/µL of SADS-CoV genomic standard recombinant plasmid in 95% of cases. The assay was performed in less than 30 min and no cross-reactions were observed with eight other common viruses that affect swine, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudo rabies virus (PRV), swine influenza virus (SIV), seneca valley virus (SVA), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). The coefficient of variation (C.V.) values of the two standards dilutions and three positive clinical sample ranged from 2.95% to 4.71%. A total of 72 clinical fecal samples from swine with diarrheal symptoms were analyzed with the developed RT-RPA and quantitative RT-PCR. There was 98.61% agreement between the RT-RPA and the quantitative real-time PCR results. CONCLUSIONS: These results indicated that the developed RT-RPA assay had good specificity, sensitivity, stability and repeatability. The study successfully established a broadly reactive RT-RPA assay for SADS-CoV detection.


Subject(s)
Alphacoronavirus , Coronavirus Infections , Nucleic Acids , Swine Diseases , Alphacoronavirus/genetics , Animals , Coronavirus Infections/diagnosis , Coronavirus Infections/veterinary , Diarrhea/diagnosis , Diarrhea/veterinary , Real-Time Polymerase Chain Reaction/veterinary , Recombinases , Sensitivity and Specificity , Swine , Swine Diseases/diagnosis
4.
Biosens Bioelectron ; 217: 114739, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-2031163

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.


Subject(s)
Biosensing Techniques , COVID-19 , Nucleic Acids , COVID-19/diagnosis , Humans , Nucleic Acid Amplification Techniques/methods , Peroxidases , RNA, Viral/analysis , RNA, Viral/genetics , Recombinases , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
Virulence ; 13(1): 1471-1485, 2022 12.
Article in English | MEDLINE | ID: covidwho-2017508

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.


Subject(s)
Nucleic Acids , Recombinases , Animals , Deltacoronavirus , Humans , Nucleic Acid Amplification Techniques , Recombinases/genetics , Recombinases/metabolism , Reproducibility of Results , Reverse Transcription , Sensitivity and Specificity , Swine
6.
Talanta ; 252:123835, 2023.
Article in English | ScienceDirect | ID: covidwho-1984108

ABSTRACT

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region “N gene.” Under isothermal reaction conditions, we integrated ligation (Lig;high selectivity) and recombinase polymerase amplification (RPA;high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)–sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus–mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

7.
Diagnostics (Basel) ; 12(5)2022 May 19.
Article in English | MEDLINE | ID: covidwho-1928513

ABSTRACT

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.

8.
Talanta ; 248: 123594, 2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-1867806

ABSTRACT

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases , SARS-CoV-2/genetics , Sensitivity and Specificity
9.
Egyptian Journal of Medical Human Genetics ; 23(1), 2022.
Article in English | EMBASE | ID: covidwho-1862175

ABSTRACT

Background: After the first case of COVID-19 being announced in China in December 2019, various diagnostic technologies have been developed at unprecedented pace with the aim of providing a basis for accurate clinical intervention. However, some assays including CRISPR-based diagnostics and loop-mediated isothermal amplification (LAMP) have been less explored. As new COVID-19 technologies emerge, there is need for them to be assessed, validated and improved upon. Moreover, there is paucity of data on the essential factors governing the selection of an appropriate diagnostic approach within the correct timeframe. Myths and origin of SARS-CoV-2 remain to be controversial. Consequently, this review aims at exploring the current COVID-19 diagnostic technologies, performance evaluation, principles, suitability, specificity, sensitivity, successes and challenges of the technologies for laboratory and bedside testing. Main Body: To date, there exist more publications on COVID-19 diagnostics as compared to the Zika virus. The SARS-CoV-2 virus genome profiles were readily available by 31st of December 2019. This was attributed to the fast-paced sharing of the epidemiological and diagnostics data of COVID-19. Timely profiling of the virus genome accelerated the development of diagnostic technologies. Furthermore, the rapid publication of studies that evaluated several diagnostic methods available provided baseline information on how the various technologies work and paved way for development of novel technologies. Conclusion: Up to date, RT-PCR is the most preferred as compared to the other assays. This is despite the repeated false negatives reported in many of the study findings. Considering that COVID-19 has caused devastating effects on the economy, healthcare systems, agriculture and culture, timely and accurate detection of the virus is paramount in the provision of targeted therapy hence reducing chances of drug resistance, increased treatment costs and morbidity. However, information on the origin of SARS-CoV-2 still remains elusive. Furthermore, knowledge and perception of the patients toward management of SARS-CoV-2 are also paramount to proper diagnosis and management of the pandemic. Future implications of the misperceptions are that they may lead to increased non-compliance to SARS-CoV-2-related World Health Organization (WHO) policies and guidelines.

10.
Zhurnal Mikrobiologii Epidemiologii i Immunobiologii ; 99(1):126-138, 2022.
Article in Russian | Scopus | ID: covidwho-1812155

ABSTRACT

In this review, methods for isothermal amplification of nucleic acids are considered and analyzed, in particular, loop isothermal amplification of DNA and RNA (LAMP/RT-LAMP), helicase-dependent amplification (HDA) and recombinase polymerase amplification (RPA). The advantages and disadvantages of each of the techniques are described. The possibility of their application in the molecular diagnostics of infectious diseases is evaluated. A brief review of the literature on the use of LAMP, HDA, RPA in the diagnostics of viral, bacterial infections and diseases of protozoal etiology was conducted. It has been shown that the LAMP method has a number of advantages over other diagnostic methods: High efficiency, specificity, simplicity, turnaround time and minimum requirements for instrument equipment. As a result, it is concluded that loop isothermal amplification is a promising method for detecting the DNA/RNA of various pathogens. The data on the introduction of the LAMP method in the diagnostics of particularly dangerous bacterial and viral infections, including for the detection of RNA of a new coronavirus infection (SARS-CoV-2) in clinical samples, are presented. © 2022, Central Research Institute for Epidemiology. All rights reserved.

11.
ACS Synth Biol ; 11(4): 1555-1567, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1773922

ABSTRACT

Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/µL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method's sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.


Subject(s)
COVID-19 , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Diagnostics (Basel) ; 12(3)2022 Mar 02.
Article in English | MEDLINE | ID: covidwho-1760429

ABSTRACT

Hepatitis B (HBV) infection is a major public health concern. Perinatal transmission of HBV from mother to child represents the main mode of transmission. Despite the existence of effective immunoprophylaxis, the preventive strategy is inefficient in neonates born to mothers with HBV viral loads above 2 × 105 IU/mL. To prevent mother-to-child transmission, it is important to identify highly viremic pregnant women and initiate antiviral therapy to decrease their viral load. We developed a simple innovative molecular approach avoiding the use of automatic devices to screen highly viremic pregnant women. This method includes rapid DNA extraction coupled with an isothermal recombinase polymerase amplification (RPA) combined with direct visual detection on a lateral flow assay (LFA). We applied our RPA-LFA approach to HBV DNA-positive plasma samples with various loads and genotypes. We designed a triage test by adapting the analytical sensitivity to the recommended therapeutic decision threshold of 2 × 105 IU/mL. The sensitivity and specificity were 98.6% (95% CI: 92.7-99.9%) and 88.2% (95% CI: 73.4-95.3%), respectively. This assay performed excellently, with an area under the ROC curve value of 0.99 (95% CI: 0.99-1.00, p < 0.001). This simple method will open new perspectives in the development of point-of-care testing to prevent HBV perinatal transmission.

13.
Biosensors (Basel) ; 12(2)2022 Jan 27.
Article in English | MEDLINE | ID: covidwho-1700284

ABSTRACT

Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft-but tight-contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.


Subject(s)
Body Temperature , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acids , Wearable Electronic Devices , COVID-19/diagnosis , DNA , Escherichia coli O157 , Humans , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/isolation & purification , Recombinases/chemistry , Recombinases/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
14.
Front Mol Biosci ; 8: 821506, 2021.
Article in English | MEDLINE | ID: covidwho-1674359

ABSTRACT

The novel coronavirus disease COVID-19 has become one of the most socially significant infections. One of the main models for COVID-19 pathogenesis study and anti-COVID-19 drug development is laboratory animals sensitive to the virus. Herein, we report SARS-CoV-2 infection in novel transgenic mice conditionally expressing human ACE2 (hACE2), with a focus on viral distribution after intranasal inoculation. Transgenic mice carrying hACE2 under the floxed STOP cassette [(hACE2-LoxP(STOP)] were mated with two types of Cre-ERT2 strains (UBC-Cre and Rosa-Cre). The resulting offspring with temporal control of transgene expression were treated with tamoxifen to induce the removal of the floxed STOP cassette, which prevented hACE2 expression. Before and after intranasal inoculation, the mice were weighed and clinically examined. On Days 5 and 10, the mice were sacrificed for isolation of internal organs and the further assessment of SARS-CoV-2 distribution. Intranasal SARS-CoV-2 inoculation in hACE2-LoxP(STOP)×UBC-Cre offspring resulted in weight loss and death in 6 out of 8 mice. Immunostaining and focus formation assays revealed the most significant viral load in the lung, brain, heart and intestine samples. In contrast, hACE2-LoxP(STOP) × Rosa-Cre offspring easily tolerated the infection, and SARS-CoV-2 was detected only in the brain and lungs, whereas other studied tissues had null or negligible levels of the virus. Histological examination revealed severe alterations in the lungs, and mild changes were observed in the brain tissues. Notably, no changes were observed in mice without tamoxifen treatment. Thus, this novel murine model with the Cre-dependent activation of hACE2 provides a useful and safe tool for COVID-19 studies.

15.
Mol Biol Rep ; 49(4): 2847-2856, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1661716

ABSTRACT

BACKGROUND: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. METHODS: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. RESULTS: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. CONCLUSIONS: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.


Subject(s)
Bacteriophage T4/enzymology , DNA, Viral/chemistry , DNA-Binding Proteins/chemistry , Membrane Proteins/chemistry , Nucleic Acid Amplification Techniques , SARS-CoV-2/chemistry , Viral Proteins/chemistry , DNA, Viral/genetics , SARS-CoV-2/genetics
16.
Open Microbiology Journal ; 15(1):77-86, 2021.
Article in English | EMBASE | ID: covidwho-1590897

ABSTRACT

Currently, there are numerous under development or developed assays with various sensitivities and specificities for diagnosis of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. The World Health Organization (WHO) has approved several detection protocols based on real-time reverse transcription PCR (RT-qPCR) and the reliability of tests to detect the N, S, or RdRp/Hel genes of the SARS-Cov-2 virus has also investigated. Among these targets, COVID-19-RdRp/Hel targets represented the highest sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has also been developed to rapidly and efficiently amplify RNA under isothermal conditions. Other isothermal amplification approaches such as nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), and rolling circle amplification (RCA) have also been reported for detecting coronaviruses but like LAMP assay. Different serological tests, including neutralization tests, immunofluorescent (IFA), enzyme-linked immunosorbent (ELISA), and western blotting assays, are available. Point-of-care tests (POCT) are emerging to detect the virus genome, IgG, or IgM antibodies against SARS-CoV-2. The advent of more sensitive, cheaper, and easier-to-perform diagnostic tests seems to be a fundamental prerequisite to improve the diagnosis of COVID-19 infection. Herein, we reviewed several commercially available diagnostic methods used in many clinical laboratories to detect COVID-19.

17.
Int J Infect Dis ; 114: 105-111, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587637

ABSTRACT

OBJECTIVES: The democratization of diagnostics is one of the key challenges towards containing the transmission of coronavirus disease 2019 (COVID-19) around the globe. The operational complexities of existing PCR-based methods, including sample transfer to advanced central laboratories with expensive equipment, limit their use in resource-limited settings. However, with the advent of isothermal technologies, the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible at decentralized facilities. METHODS: In this study, two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples. A total of 76 real-time reverse transcription PCR (real-time RT-PCR) confirmed COVID-19 cases and 100 negative controls were evaluated to determine the diagnostic performance of the isothermal methods. RESULTS: This investigation revealed equally promising diagnostic accuracy of the two methods, with a sensitivity of 76.32% (95% confidence interval 65.18-85.32%) when the target genes were RdRP and ORF1ab for RT-RPA and RT-RAA, respectively; the combination of N and RdRP in RT-RPA augmented the accuracy of the assay at a sensitivity of 85.53% (95% confidence interval 75.58-92.55%). Furthermore, high specificity was observed for each of the methods, ranging from 94.00% to 98.00% (95% confidence interval 87.40-9.76%). CONCLUSIONS: Considering the diagnostic accuracies, both RT-RPA and RT-RAA appear to be suitable assays for point-of-need deployment for the detection of the pathogen, understanding its epidemiology, case management, and curbing transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases/metabolism , Reverse Transcription , Sensitivity and Specificity
18.
Pharmaceutics ; 13(11)2021 Nov 12.
Article in English | MEDLINE | ID: covidwho-1538442

ABSTRACT

Messenger RNA (mRNA) has generated great attention due to its broad potential therapeutic applications, including vaccines, protein replacement therapy, and immunotherapy. Compared to other nucleic acids (e.g., siRNA and pDNA), there are more opportunities to improve the delivery efficacy of mRNA through systematic optimization. In this report, we studied a high-throughput library of 1200 functional polyesters for systemic mRNA delivery. We focused on the chemical investigation of hydrophobic optimization as a method to adjust mRNA polyplex stability, diameter, pKa, and efficacy. Focusing on a region of the library heatmap (PE4K-A17), we further explored the delivery of luciferase mRNA to IGROV1 ovarian cancer cells in vitro and to C57BL/6 mice in vivo following intravenous administration. PE4K-A17-0.2C8 was identified as an efficacious carrier for delivering mRNA to mouse lungs. The delivery selectivity between organs (lungs versus spleen) was found to be tunable through chemical modification of polyesters (both alkyl chain length and molar ratio in the formulation). Cre recombinase mRNA was delivered to the Lox-stop-lox tdTomato mouse model to study potential application in gene editing. Overall, we identified a series of polymer-mRNA polyplexes stabilized with Pluronic F-127 for safe and effective delivery to mouse lungs and spleens. Structure-activity relationships between alkyl side chains and in vivo delivery were elucidated, which may be informative for the continued development of polymer-based mRNA delivery.

19.
Biosens Bioelectron ; 197: 113771, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1536447

ABSTRACT

Due to the similar clinical symptoms of influenza (Flu) and coronavirus disease 2019 (COVID-19), there is a looming infection threat of concurrent Flu viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we introduce a customized isothermal amplification integrated lateral flow strip (LFS) that is capable performing duplex reverse transcription-recombinase polymerase amplification (RT-RPA) and colorimetric LFS in a sequential manner. With customized amplification primer sets targeted to SARS-CoV-2 (opening reading frame 1a/b and nucleoprotein genes) and Flu viruses (Flu A and Flu B), the platform allows the rapid and simultaneous visual screening of SARS-CoV-2 and Flu viruses (Flu A and Flu B) without cross reactivity, false positives, and false negatives. Moreover, it maximally eases the detection, reduces the detection time (1 h), and improves the assay performance to detect as low as 10 copies of the viral RNA. Its clinical application is powerfully demonstrated with 100% accuracy for evaluating 15 SARS-CoV-2-positive clinical samples, 10 Flu viruses-positive clinical samples, and 5 negative clinical samples, which were pre-confirmed by standard qRT-PCR. We envision this portable device can meet the increasing need of online monitoring the serious infectious diseases that substantially affects health care systems worldwide.


Subject(s)
Biosensing Techniques , COVID-19 , Influenza, Human , Humans , Influenza, Human/diagnosis , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Sensitivity and Specificity
20.
China CDC Wkly ; 3(46): 973-976, 2021 Nov 12.
Article in English | MEDLINE | ID: covidwho-1513533

ABSTRACT

INTRODUCTION: The best approach to preventing the importation of coronavirus disease 2019 (COVID-19) is enhancing the detection capacity at customs. The rapid detection is of utmost importance and therefore highly demanded. METHODS: We conducted a field validation study of a duplex real-time reverse transcription recombinase-aided amplification (RT-RAA) assay in Zhoushan and Hangzhou customs, in Zhejiang Province, China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay kit routinely used at customs was used in parallel, and the duration the two methods took to complete a specific number of samples was compared. RESULTS: Among 506 samples collected, RT-RAA results were consistent with the RT-PCR results. The sensitivity and specificity were 100%, the total coincidence rate was 100%, and the Kappa value was 1 (P<0.05) for both methods. The RT-RAA kit took a significantly shorter time in testing the 20-200 samples than the RT-PCR kit. DISCUSSION: The RT-RAA detection method is more efficient and suitable for use at customs than RT-PCR assay to realize rapid customs clearance of 200 or fewer samples.

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