BACKGROUND: Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management. Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention. Quantum dot-encoded microspheres have been widely used in immunodetection. The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis. Thus, establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis.
OBJECTIVE: To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology, which forms the foundation for the assays of multiple respiratory virus biomarkers.
METHODS: Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B. The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer, and the detection conditions were optimized to establish the influenza A and B antigen codetection method, which was utilized for their detection in clinical samples. The results were compared with the fluorescence quantitative polymerase chain reaction (PCR) method to validate the clinical performance of this method.
RESULTS: The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens, respectively, which both ranged from 15.6 to 250000 pg/mL. In the clinical sample evaluation, the proposed method well correlated with the fluorescent quantitative PCR method, with positive, negative, and overall compliance rates of 57.4%, 100%, and 71.6%, respectively.
CONCLUSIONS: A multiplex assay for quantitative detection of influenza A and B virus antigens has been established, which is characterized by high sensitivity, good specificity, and a wide detection range and is promising for clinical applications.

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5\'-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

BACKGROUND: Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), pose a significant threat to public health. Existing detection methods, like cultivation-based techniques, demand significant time and labor, while molecular diagnostic techniques, such as PCR, necessitate sophisticated instrumentation and skilled personnel. Although previous multiplex loop-mediated isothermal amplification assays based on fluorescent dyes (mfLAMP) offer simplicity and cost-effectiveness, they are prone to false-positive results. Therefore, developing a rapid and efficient multiplex assay for high-sensitivity MRSA is imperative to create a practical diagnostic tool for point-of-care testing.
RESULTS: Here, we developed a mfLAMP combined with a lateral flow assay (mfLAMP-LFA) for the visual and simultaneous detection of the mecA (PBP2a-specific marker) and nuc (S. aureus-specific marker) genes in MRSA. We optimized mfLAMP-LFA using graphene oxide (GO)-based purification and specific DNA probes and evaluated its sensitivity, specificity, and stability. Utilizing GO to mitigate false-positive results by acting as a trap for free DNA probes, the mfLAMP-LFA method successfully identified mecAf and nucf-probes, exhibiting distinct red, green, and yellow fluorescence signals. The detection sensitivity of the developed mfLAMP-LFA method (1 CFU mL-1 in phosphate-buffered saline (PBS)) was comparable to other highly sensitive MRSA detection methods (1 CFU mL-1 in PBS). Furthermore, the method demonstrated specificity for MRSA, detecting it in irrigation water samples within the desired range and achieving reliable recovery rates from spiked samples.
CONCLUSIONS: This novel strategy is the first to incorporate GO into mfLAMP-LFA, enabling specific and sensitive MRSA detection and advancing rapid bacterial detection. This assay facilitates MRSA diagnostics, contributing to improved public health and food safety by delivering rapid, cost-effective point-of-care results. It enables the simultaneous detection of multiple bacteria, even in irrigation water samples artificially inoculated with MRSA, which contain aerobic bacteria at 2.7 × 102 CFU mL-1.

A novel electrochemical aptasensor was prepared for the simultaneous determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA). Composites of Au nanoparticles and polyethyleneimine-reduced graphene oxide (AuNPs/PEI-RGO) with good electrical conductivity and high specific surface area were employed as the supporting substrate, demonstrating the ability to provide more binding sites for aptamers and accelerate the electron transfer. Aptamers were immobilized on a AuNPs/PEI-RGO surface to specifically recognize AFB1 and OTA. A metal-organic framework of UiO-66-NH2 served as the signal carrier to load metal ions of Cu2+ and Pb2+, which facilitated the generation of independent current peaks and effectively improved the electrochemical signals. The prepared aptasensor exhibited sensitive current responses for AFB1 and OTA with a linear range of 0.01 to 1000 ng/mL, with detection limits of 6.2 ng/L for AFB1 and 3.7 ng/L for OTA, respectively. The aptasensor was applied to detect AFB1 and OTA in cereal samples, achieving results comparable with HPLC-MS, with recovery results from 92.5% to 104.1%. With these merits of high sensitivity and good selectivity and stability, the prepared aptasensor proved to be a powerful tool for evaluating contaminated cereals.

In this work, a series of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) radicals bearing different functional groups were exploited as a simple catalyst to promote electrochemiluminescence (ECL) generation in luminol/H2O2 system. These TEMPO radicals were found to facilitate the electrochemical oxidation of H2O2 and luminol through different catalytic mechanisms, as well as the subsequent ECL generation of luminol/H2O2 system. The electrochemical oxidation and luminol ECL generation could be tuned by the functional group on the para-position of TEMPO, for which the structure/activity relationship was revealed. Finally, with the combination of enzymatic system, luminol ECL enhancement up to 9.6-fold was obtained through the catalysis of 4-hydroxyl-TEMPO. The enhanced luminol ECL allows acquiring brighter ECL images in a single-electrochemical system (SEES) for multiplex detection of cholesterol, H2O2 and glucose.

Biomarkers screening is a benefit approach for early diagnosis of major diseases. In this study, magnetic nanoparticles (MNPs) have been utilized as labels to establish a multi-line immunochromatography (MNP-MLIC) for simultaneous detection of carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 19-9), and alpha-fetoprotein (AFP) in a single serum sample. Under the optimal parameters, the three biomarkers can be rapidly and simultaneously qualitative screening within 15 min by naked eye. As for quantitative detection, the MNP-MLIC test strips were precisely positioned and captured by a smartphone, and signals on the test and control lines were extracted by ImageJ software. The signal ratio of test and control lines has been calculated and used to plot quantitative standard curves with the logarithmic concentration, of which the correlation coefficients are more than 0.99, and the limit of detection for CEA, CA 19-9, and AFP were 0.60 ng/mL, 1.21 U/mL, and 0.93 ng/mL, respectively. The recoveries of blank serum were 75.0 ~ 112.5% with the relative standard deviation ranging from 2.5 to 15.3%, and the specificity investigation demonstrated that the MNP-MLIC is highly specific to the three biomarkers. In conclusion, the developed MNP-MLIC offers a rapid, simple, accurate, and highly specific method for simultaneously detecting multiple biomarkers in serum samples, which provides an efficient and accurate approach for the early diagnosis of diseases.

Introduction: Nucleic acid tests for blood donor screening have improved the safety of the blood supply; however, increasing numbers of emerging pathogen tests are burdensome. Multiplex testing platforms are a potential solution. Methods: The Blood Borne Pathogen Resequencing Microarray Expanded (BBP-RMAv.2) can perform multiplex detection and identification of 80 viruses, bacteria and parasites. This study evaluated pathogen detection in human blood or plasma. Samples spiked with selected pathogens, each with one of 6 viruses, 2 bacteria and 5 protozoans were tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of primers, and hybridized to a microarray. The reported sequences were aligned to a database to identify the pathogen. To directly compare the microarray to an emerging molecular approach, the amplified nucleic acids were also submitted to nanopore next generation sequencing (NGS). Results: The BBP-RMAv.2 detected viral pathogens at a concentration as low as 100 copies/ml and a range of concentrations from 1,000 to 100,000 copies/ml for all the spiked pathogens. Coded specimens were identified correctly demonstrating the effectiveness of the platform. The nanopore sequencing correctly identified most samples and the results of the two platforms were compared. Discussion: These results indicated that the BBP-RMAv.2 could be employed for multiplex detection with potential for use in blood safety or disease diagnosis. The NGS was nearly as effective at identifying pathogens in blood and performed better than BBP-RMAv.2 at identifying pathogen-negative samples.

Current quantitative gene expression detection in genomic and transcriptomic research heavily relies on quantitative real-time PCR (qPCR). While existing multiplex gene detection techniques offer simultaneous analysis of multiple targets, we present an alternative assay capable of detecting gene expression simultaneously within a single well. This highly sensitive method utilizes πCode MicroDiscs, featuring unique identification patterns and fluorescent detection. Our study compared this multiplex πCode platform with a qPCR platform for profiling cytokine gene expression. The πCode MicroDisc assay successfully demonstrated the expression of polymerization markers for M1- and M2-like macrophages generated from THP-1-derived macrophages in a qualitative assay. Additionally, our findings suggest a pattern agreement between the πCode assay and the qPCR assay, indicating the potential of the πCode technology for comparative gene expression analysis. Regarding the inherent sensitivity and linearity, the developed πCode assay primarily provides qualitative gene expression to discriminate the polarization of macrophages. This remarkable capability presents substantial advantages for researchers, rendering the technology highly suitable for high-throughput applications in clinical diagnosis and disease monitoring.

Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/μL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/μL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.