Clinical Swab Samples Research Articles

Detection of OXA-23, OXA-48, OmpA and bla-TEM genes in carbapenem-resistance Acinetobacter baumannii and biofilm formation in burn patients at Al-Anbar Government

Background. Antibiotic-resistant bacteria represent a highly global health issue by causing high rates of morbidity and mortality in burn units, whereas biofilms significantly contribute to complications in burn patients. Objectives. The study investigates the prevalence of (OXA-23, OXA-48, OmpA and bla-TEM) and biofilm formation of Acinetobacter baumannii in the Burn Unit of the Al-Anbar government. Methods. A total of 120 clinical swab samples from burn patient were collected from December 2023 to April 2024 from the burn units of Ramadi Teaching Hospital and Falluja Teaching Hospital for the study. The colonies were identified using conventional and Vitek methods. Colorimetric technique (MTP) and multiplex PCR were used to detect biofilms and resistance genes. Results. Among 120 samples, 40 (33.3%) were Acinetobacter baumannii, 34 (80%) were MDR- resistant, and 6 (15%) were XDR. 90% of the isolates were resistant to Cefotaxime, and Colistin was more sensitive in all forty isolates. All 40 isolates formed significant biofilms. The percentage of positive results for OXA-23, OmpA and bla-TEM genes were 80%, 65%, and 55% of respectively. Carbapenem testing is essential to identify carbapenem-producing strains from non-producing strains due to the high prevalence of multidrug-resistant A. baumannii with the OXA-23 and OmpA genes. Conclusion. These findings highlight the urgent need for effective infection control measures and the development of new therapeutic strategies to manage these infections in burn patients.

Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection.

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Hermetic microfluidic device for point-of-care viral nucleic acid testing

There is an unmet clinical and public health need for rapid and accurate point-of-care (POC) diagnosis and management of infectious pathogens, especially in resource-limited environments. In this work, we present a microfluidic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) rapid test (μDART) device for POC detection of various viral pathogens. This platform hermetically integrates the modules of extraction-free sample lysis, loop-mediated isothermal amplification (LAMP) with lyophilized reagent beads, and real-time colorimetric signal sensing to realize "sample-in and result-out" in 15–30 min. Test results can be sent to a mobile phone for remote monitoring. Automated high-precision liquid handling is achieved by a combination of negative-pressure-driven microchambers and self-sealing valves, thus simplifying manual operations and eliminating the leakage of nucleic acid aerosols. The limit of detection (LOD) of the device is as low as ∼53 copies/mL or ∼2 copies/reaction for SARS-CoV-2, which is as sensitive as the gold standard of nucleic acid testing. To validate our device’s clinical applications, we tested both DNA and RNA viruses as representative pathogens including 537 clinical nasal swab samples for SARS-CoV-2, 74 cervical swab samples for human papillomavirus 16/18 (HPV16/18) and 82 pet swab samples for feline cupripoxvirus (FCV) and feline herpesvirus (FHV). Across these tests, the clinical sensitivity and specificity were 92.5% - 97.7% and 99.7%-100%, respectively. We demonstrate that the μDART is able to provide a quick and accurate result for POCT and self-testing, and can connect to health care wirelessly to achieve remote monitoring.

Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae

BackgroundCurable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. MethodsWe identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/μL to 1 × 10−3 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. ResultsOur newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL. ConclusionTherefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Combination-Lock SlipChip Integrating Nucleic Acid Sample Preparation and Isothermal LAMP Amplification for the Detection of SARS-CoV-2.

Nucleic acid analysis with an easy-to-use workflow, high specificity and sensitivity, independence of sophisticated instruments, and accessibility outside of the laboratory is highly desirable for the detection and monitoring of infectious diseases. Integration of laboratory-quality sample preparation on a hand-held system is critical for performance. A SlipChip device inspired by the combination lock can perform magnetic bead-based nucleic acid extraction with several clockwise and counterclockwise rotations. A palm-sized base station was developed to assist sample preparation and provide thermal control of isothermal nucleic acid amplification without plug-in power. The loop-mediated isothermal amplification reaction can be performed with a colorimetric method and directly analyzed by the naked eye or with a mobile phone app. This system achieves good bead recovery during the sample preparation workflow and has minimal residue carryover from the lysis and elution buffers. Its performance is comparable to that of the standard laboratory protocol with real-time qPCR amplification methods. The entire workflow is completed in less than 35 min and the device can achieve 500 copies/mL sensitivity. Thirty clinical nasal swab samples were collected and tested with a sensitivity of 95% and a specificity of 100% for SARS-CoV-2. This combination-lock SlipChip provides a promising fast, easy-to-use nucleic acid test with bead-based sample preparation that produces laboratory-quality results for point-of-care settings, especially in home use applications.

Optimization of Loop-Mediated Isothermal Amplification Reaction for Detecting Streptococcus pneumoniae Serotypes in Clinical Nasopharynx Swab Samples

Current molecular PCR-based methods for identifying Streptococcus pneumoniae, the primary cause of pneumonia, meningitis, and other invasive diseases, are accurate but require expensive infrastructure and have a long run time, which restricts their use, particularly in developing countries. LAMP, or loop-mediated isothermal amplification, is a low-cost alternative to polymerase chain reaction (PCR) that can be used quickly because the reaction only takes place at a constant temperature. We aimed to develop a LAMP assay for rapid detection of Streptococcus pneumoniae serotypes in nasopharynx swab samples. The LAMP primers were designed using a conserved region of the lytA gene. An incubation time range of 30 to 60 minutes was studied to optimize the LAMP reaction. The real-time fluorescence intensity was monitored during the amplification reaction. Clinical nasopharynx swab samples identified as Streptococcus pneumoniae and their serotypes were tested to evaluate the performance of LAMP. To investigate the specificity of the LAMP, Streptococcus species samples and non-Streptococcus species samples were analyzed. In conclusion, the optimized LAMP assay is capable of detecting Streptococcus pneumoniae and its serotypes in nasopharynx swab samples.

A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus.

Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously. The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control. RSV and ADV could be detected at levels as low as 104 copies/mL and 103 copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency. This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.

A portable all-in-one microfluidic device with real-time colorimetric LAMP for HPV16 and HPV18 DNA point-of-care testing

Screening for high-risk human papillomavirus (HPV) infection is one of the most important preventative measures for cervical cancer. However, fast, convenient, and low-cost HPV detection remains challenging, especially in resource-limited settings. Here, we report a portable all-in-one device (PAD) for point-of-care testing (POCT) for HPV16 and HPV18 DNA in cervical swabs. The PAD was engineered to integrate modules for extraction-free sample lysis, loop-mediated isothermal amplification (LAMP) with lyophilized reagent beads, and real-time colorimetric signal sensing into a single miniaturized device, considerably shortening the sample-to-result time to 15 min. The precision liquid handling in the completely sealed microfluidic chip is achieved by a uniquely designed pressure-balanced automatic liquid flow mechanism, thereby eliminating the need for manual manipulation of liquids and thus the risk of biohazards. The PAD employs an improved real-time colorimetric LAMP (rcLAMP) assay with a limit of detection (LOD) of 1 copy/μL, enabled by enhanced assay chemistry to maximize the reaction kinetics. To validate this device for clinical application, we tested 206 clinical cervical swab samples and obtained a sensitivity of 92.1% and a specificity of 99.0%. This custom PAD enabled by microfluidic and electronic engineering techniques can be configured for the simultaneous detection of HPV16 and HPV18 or other pathogens in point-of-care applications.

Development of a one-step multiplex (RT)-PCR for simultaneous detection of virus-induced respiratory disease complex in dogs in Vietnam

Canine infectious respiratory disease (CIRD) viruses, particularly canine distemper virus (CDV), canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), are the primary agents responsible for respiratory diseases in dogs. A dog can be infected with a single or multiple viruses with similar clinical signs. Although CIRD is globally prevalent in dogs, investigation into the causative viruses in Vietnam remains limited. This study aims to identify the presence of CIRD virus(es) in dogs via molecular detection. Three primer sets were newly designed and applied into (RT)-PCR reaction to detect viral genes of CDV, CAV-2, and CPIV in a commercial vaccine (Vanguard® Plus 5/L, Zoetis, USA). Both simplex and multiplex (RT)-PCR reactions using the three primer sets could detect the presence of CAV-2, CDV, and CPIV in the vaccine sample. Subsequently, the applicability of the one-step multiplex (RT)-PCR was demonstrated to test for 27 clinical nasal swab samples collected from dogs suspected of having CIRD. The results showed that our (RT)-PCR could detect CIRD virus(es) in all tested clinical samples. PCR amplicons for each representative CAV-2, CDV, and CPIV were selected for sequencing and showed high genetic similarity to respective field viruses. In conclusion, this study successfully developed a one-step multiplex (RT)-PCR reaction to detect and differentiate causative viruses of CIRD in dogs.

Gravity-driven flow control in a fully integrated microfluidic cartridge for molecular point-of-care testing.

Molecular point-of-care testing (POCT) system is crucial for the timely prevention and control of infectious diseases. We recently proposed a gravity-driven microfluidic cartridge for molecular POCT detection, without the need for external sources or actuators, demonstrating the advantages in terms of the reduced cartridge size and low development costs. How to achieve precise control of liquid flow behavior is challenging for the gravity-driven cartridge. In this work, we explored the underlying mechanism of flow control in the cartridge and offered optimized solutions for our cartridge design to achieve precise control of dynamic flow rates and enhance pumping efficiency significantly. Through the computational fluid dynamics simulations, we demonstrated that adopting an asymptotic contraction chamber geometry design and a closed-loop air flow channel design with the cartridge inlet can facilitate stable laminar flow of the liquid in our microfluidic cartridge, enabling precise control of flow velocity. We further optimized the microchannel diameter and the contact angle of the liquid with the microchannel wall. The effectiveness of the optimized cartridge for POCT detection was well validated by the accurate detection of the human papillomavirus type 16 virus in the 120 clinical swab samples.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics.

Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, andHPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention. KEY POINTS: • The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes. • A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity. • The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.

Development of a loop-mediated isothermal amplification (LAMP)-based electrochemical test for rapid detection of SARS-CoV-2

Rapid, reliable, sensitive, portable, and accurate diagnostics are required to control disease outbreaks such as COVID-19 that pose an immense burden on human health and the global economy. Here we developed a loop-mediated isothermal amplification (LAMP)-based electrochemical test for the detection of SARS-CoV-2 that causes COVID-19. The test is based on the oxidation-reduction reaction between pyrophosphates (generated from positive LAMP reaction) and molybdate that is detected by cyclic voltammetry using inexpensive and disposable carbon screen printed electrodes. Our test showed higher sensitivity (detecting as low as 5.29 RNA copies/μL) compared to the conventional fluorescent reverse transcriptase (RT)-LAMP. We validated our tests using human serum and saliva spiked with SARS-CoV-2 RNA and clinical (saliva and nasal-pharyngeal) swab samples demonstrating 100% specificity and 93.33% sensitivity. Our assay provides a rapid, specific, and sensitive test with an electrochemical readout in less than 45min that could be adapted for point-of-care settings.

Culture-Independent Workflow for Nanopore MinION-Based Sequencing of Influenza A Virus.

Whole-genome sequencing (WGS) of influenza A virus (IAV) is crucial for identifying diverse subtypes and newly evolved variants and for selecting vaccine strains. In developing countries, where facilities are often inadequate, WGS is challenging to perform using conventional next-generation sequencers. In this study, we established a culture-independent, high-throughput native barcode amplicon sequencing workflow that can sequence all influenza subtypes directly from a clinical specimen. All segments of IAV in 19 clinical specimens, irrespective of their subtypes, were amplified simultaneously using a two-step reverse transcriptase PCR (RT-PCR) system. First, the library was prepared using the ligation sequencing kit, barcoded individually using the native barcodes, and sequenced on the MinION MK 1C platform with real-time base-calling. Then, subsequent data analyses were performed with the appropriate tools. WGS of 19 IAV-positive clinical samples was carried out successfully with 100% coverage and 3,975-fold mean coverage for all segments. This easy-to-install and low-cost capacity-building protocol took only 24 h complete from extracting RNA to obtaining finished sequences. Overall, we developed a high-throughput portable sequencing workflow ideal for resource-limited clinical settings, aiding in real-time surveillance, outbreak investigation, and the detection of emerging viruses and genetic reassortment events. However, further evaluation is required to compare its accuracy with other high-throughput sequencing technologies to validate the widespread application of these findings, including WGS from environmental samples. IMPORTANCE The Nanopore MinION-based influenza sequencing approach we are proposing makes it possible to sequence the influenza A virus, irrespective of its diverse serotypes, directly from clinical and environmental swab samples, so that we are not limited to virus culture. This third-generation, portable, multiplexing, and real-time sequencing strategy is highly convenient for local sequencing, particularly in low- and middle-income countries like Bangladesh. Furthermore, the cost-efficient sequencing method could provide new opportunities to respond to the early phase of an influenza pandemic and enable the timely detection of the emerging subtypes in clinical samples. Here, we meticulously described the entire process that might help the researcher who will follow this methodology in the future. Our findings suggest that this proposed method is ideal for clinical and academic settings and will aid in real-time surveillance and in the detection of potential outbreak agents and newly evolved viruses.

Facile and Unplugged Surface Plasmon Resonance Biosensor with NIR-Emitting Perovskite Nanocomposites for Fast Detection of SARS-CoV-2.

The emergence of the coronavirus disease 2019 (COVID-19) pandemic prompted researchers to develop portable biosensing platforms, anticipating to detect the analyte in a label-free, direct, and simple manner, for deploying on site to prevent the spread of the infectious disease. Herein, we developed a facile wavelength-based SPR sensor built with the aid of a 3D printing technology and synthesized air-stable NIR-emitting perovskite nanocomposites as the light source. The simple synthesis processes for the perovskite quantum dots enabled low-cost and large-area production and good emission stability. The integration of the two technologies enabled the proposed SPR sensor to exhibit the characteristics of lightweight, compactness, and being without a plug, just fitting the requirements of on-site detection. Experimentally, the detection limit of the proposed NIR SPR biosensor for refractive index change reached the 10-6 RIU level, comparable with that of state-of-the-art portable SPR sensors. In addition, the bio-applicability of the platform was validated by incorporating a homemade high-affinity polyclonal antibody toward the SARS-CoV-2 spike protein. The results demonstrated that the proposed system was capable of discriminating between clinical swab samples collected from COVID-19 patients and healthy subjects because the used polyclonal antibody exhibited high specificity against SARS-CoV-2. Most importantly, the whole measurement process not only took less than 15 min but also needed no complex procedures or multiple reagents. We believe that the findings disclosed in this work can open an avenue in the field of on-site detection for highly pathogenic viruses.

A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel

The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.