Characterizing a visual lateral flow device for rapid SARS-CoV-2 virus protein detection: pre-clinical and system assessment.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections have affected more than 769 million individuals worldwide over the last few years. Although the pandemic is transitioning into an endemic, the COVID-19 outbreak is still a global concern. A rapid screening platform is needed for effective preventive and control measures. Herein, a visual rapid lateral flow platform for SARS-CoV-2 nucleocapsid protein detection is developed. Under optimal conditions, the system demonstrated good detection sensitivity and selectivity against tested respiratory viruses. The system provides direct visual detection with a limit of 0.7 ng of the nucleocapsid protein per mL of a sample (0.7 ng mL-1) within 15 minutes. Further, a correlation between direct visual detection and semi-quantitative analysis using a reader showed a similar detection limit (R2 = 0.9571). The repeatability and reproducibility studies highlighted the potential of the system for the rapid screening of SARS-CoV-2 infection, with variations within 5% and 10% at high and low protein concentrations, respectively. Subsequent pre-clinical validation to correlate the performance with the standard molecular approach (RT-PCR) using 170 nasopharyngeal swabs demonstrated 98% estimated sensitivity (95% CI, 89.35-99.95%) and 100% specificity (95% CI, 96.38-100%). The positive and negative predictive values were reported to be 100% and 99%, respectively, with an accuracy of 99.3%. With high viral load samples (Ct value ≤25, n = 47), the system demonstrated 100% detection sensitivity and specificity. The proposed technique provides a valuable platform for potential use in rapid screening, particularly during pandemics, where diagnostic capacity and mass screening are crucial.

A simple fluorescence-based lateral flow test platform for rapid influenza B virus screening.

A simple fluorescence-based lateral flow test platform for rapid influenza B virus screening as a model target molecule was successfully developed. In this work, Cy5-loaded silica nanoparticles were directly conjugated to monoclonal antibodies, specific to the influenza B nucleoprotein, via a direct physisorption method and used as detector probes. Using this approach, the signal response to the detection was further determined using a fluorescent signal intensity measurement method via a portable reader, in combination with fluorescence imaging analysis. The degree to which the fluorescence signal response is detected is proportional to the amount of the target virus protein present in the system, reflected by the accumulation of the formed particle-antibody conjugates within the test system. Under optimized conditions, the system is capable of detecting the influenza B virus protein at a level of 0.55 µg per test within 30 min, using small sample volumes as low as 100 µL (R2 = 0.9544). In addition to its simplicity, further application of the system in detecting the influenza B virus protein was demonstrated using the viral transport media as specimen matrices. It was also shown that the system can perform the detection without cross-reactivity to other closely related respiratory viruses.

Pre-clinically evaluated visual lateral flow platform using influenza A and B nucleoprotein as a model and its potential applications.

A visual colorimetric rapid screening system based on a lateral flow device for simultaneous detection and differentiation between influenza A and B nucleoprotein as a model was developed. Monoclonal antibodies, specific for either influenza A or B nucleoproteins, were evaluated for their reactivities and were used as targeting ligands. With the best antibody pairs selected, the system exhibited good specificity to both viruses without cross reactivity to other closely related respiratory viruses. Further semi-quantitative analysis using a strip reader revealed that the system is capable of detecting influenza A and B protein content as low as 0.04 and 1 ng per test, respectively, using a sample volume as low as 100 µL, within 10 minutes (R 2 = 0.9652 and 0.9718). With a performance comparison to the commercial tests, the system demonstrated a four-to-eight-fold higher sensitivity. Pre-clinical evaluation with 101 nasopharyngeal swabs reveals correlated results with a standard molecular approach, with 89% and 83% sensitivity towards influenza A and B viruses, and 100% specificity for both viruses.

Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay.

Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.

Simultaneous discrimination and detection of influenza A(H1N1)pdm09 and seasonal influenza A viruses using a rapid immunogold biosensor.

A rapid immunogold biosensor for the simultaneous discrimination of influenza A(H1N1)pdm09 and seasonal influenza A viruses was developed successfully. Monoclonal antibodies (mAbs) that were specific for the hemagglutinin protein of the A(H1N1)pdm09 virus were produced, and the best mAb pairs were selected. Using an mAb that was specific for the influenza A nucleoprotein, a rapid immunogold biosensor for the discrimination and detection of A(H1N1)pdm09/seasonal influenza viruses was developed. When tested with 72 virus isolates, the system achieved 100 % detection of the A(H1N1)pdm09 virus without cross-reactivity against seasonal influenza A (H1, H3 subtypes) and B viruses, parainfluenza viruses, respiratory syncytial viruses, and adenoviruses. The detection limits for A(H1N1)pdm09 and seasonal strains were 5 × 10(2)-7.5 × 10(3) and 1 × 10(3)-7.5 × 10(5) TCID50/mL, respectively. When tested with 49 clinical specimens, the specificity was high (100 %). The sensitivity for the detection of A(H1N1)pdm09 and seasonal strains was 90 % and 100 %, respectively, which correlated with the results of real-time reverse transcription polymerase chain reaction as a reference method. The ability of the system to detect and discriminate the A(H1N1)pdm09 strain from the seasonal strains suggests that this method may be beneficial for investigation of outbreaks and diagnostic applications. Furthermore, this method might be a useful platform for developing a rapid diagnostic system for the simultaneous discrimination of other influenza virus subtypes during future outbreaks.

Improved sensitivity of influenza A antigen detection using a combined NP, M, and NS1 sandwich ELISA.

A new modified triple-antigen detection test was developed for the direct detection of the influenza A virus. The nucleoprotein (NP), matrix (M), and non-structural (NS1) proteins were used as target antigens because they are abundant in infected cells. Monoclonal antibodies specific to the NP, M, and NS1 proteins were generated. The antibody pairs were selected and evaluated for their reactivity individually and in combination in the triple-antigen detection using sandwich ELISA. Triple-antigen detection demonstrated a higher sensitivity than individual antigen detection when tested with both the H1N1 and H3N2 influenza A viruses. This was illustrated by the 4-fold lower limit of detection of the triple-antigen test than the individual antigen detection test. The findings demonstrated that the sensitivity of influenza A antigen detection was improved with the triple-antigen detection system as compared to individual antigen detection. Therefore, this technique could be a useful tool for the direct detection of cell-associated influenza A antigen. Furthermore, it could provide a basis for the development of a rapid triple-antigen test for influenza A diagnosis.