A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Medical diagnostic value of digital PCR (dPCR): A systematic review

Digital polymerase chain reaction (dPCR) is an emerging technique for the absolute quantification of target nucleic acids. dPCR got attention as a precise quantification tool in preclinical research, particularly when used to detect genetic mutations and result in highly precise measurements. In dPCR, the statistic of Poisson distribution was followed for the random distribution of molecules in different partitions, which is essential for dPCR quantification. Amplified target sequences in different partitions are identified by fluorescence and each partition functions as a separate PCR microreactor. Without the need for calibration, the percentage of PCR-positive partitions is sufficient to estimate the concentration of the target sequence. The present revolution in digital quantification was made possible by advancements in microfluidics, which provided effective partitioning techniques. In this paper, the contrast of the underlying ideas of quantitative real-time PCR with dPCR for the measurement of nucleic acids quantity Polymerase chain reaction (q-PCR). This review study briefly introduced the background of dPCR and compared different types of PCR, particularly the quantity of real-time qPCR and digital PCR. The fundamental concept of dPCR is also explained and also briefly compares the advantages of dPCR over qPCR and analyzes the applications of dPCR as a diagnostic tool for cancer and different types of viral species.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Rapid and Accurate Detection of SARS-CoV-2 Using an iPad-Controlled, High-Throughput, Portable, and Multiplex Hive-Chip Platform (HiCube).

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most effective measures to control the coronavirus disease 2019 (COVID-19) pandemic. However, there is still lack of an ideal detection platform capable of high sample throughput, portability, and multiplicity. Herein, by combining Hive-Chip (capillary microarray) and reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), we developed an iPad-controlled, high-throughput (48 samples at one run), portable (smaller than a backpack), multiplex (monitoring 8 gene fragments in one reaction), and real-time detection platform for SARS-CoV-2 detection. This platform is composed of a portable Hive-Chip device (HiCube; 32.7 × 29.7 × 20 cm, 5 kg), custom-designed software, and optimized Hive-Chips. RT-LAMP primers targeting seven SARS-CoV-2 genes (S, E, M, N, ORF1ab, ORF3a, and ORF7a) and one positive control (human RNase P) were designed and prefixed in the Hive-Chip. On-chip RT-LAMP showed that the limit of detection (LOD) of SARS-CoV-2 synthetic RNAs is 1 copy/µL, and there is no cross-reaction among different target genes. The platform was validated by 100 clinical samples of SARS-CoV-2, and the results were highly consistent with those of the traditional real-time PCR assay. In addition, on-chip detection of 6 other respiratory pathogens showed no cross-reactivity. Overall, our platform has great potential for fast, accurate, and on-site detection of SARS-CoV-2.

An integrated fluorescent lateral flow assay for multiplex point-of-care detection of four respiratory viruses.

This work established a highly sensitive and specific quantum dot nanobeads-based lateral flow assay for multiplex detection of four respiratory virus markers at point of care. The respiratory virus antigens were detected by fluorescent lateral flow strips within 20 min. The limits of detection for SARS-CoV-2 antigen, IAV antigen, IBV antigen, and ADV antigen were 0.01 ng/mL, 0.05 ng/mL, 0.31 ng/mL, and 0.40 ng/mL, respectively, which were superior to that of conventional AuNPs-based colorimetric lateral flow assay. The coefficients of variation of the test strip were 6.09%, 2.24%, 7.92%, and 12.43% for these four antigens, which indicated that the proposed method had good repeatability. The specificity of the detection system was verified by different combinations of these four respiratory viruses and several other respiratory pathogens. These results indicated that this method could simultaneously detect SARS-CoV-2, IAV, IBV and ADV in a short assay time, showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

Multiplex Detection of Antibody Landscapes to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/Influenza/Common Human Coronaviruses Following Vaccination or Infection With SARS-CoV-2 and Influenza.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to co-circulate, representing 2 major public health threats from respiratory infections with similar clinical presentations. SARS-CoV-2 and influenza vaccines can also now be co-administered. However, data on antibody responses to SARS-CoV-2 and influenza coinfection and vaccine co-administration remain limited. METHODS: We developed a 41-plex antibody immunity assay that can simultaneously characterize antibody landscapes to SARS-CoV-2/influenza/common human coronaviruses. We analyzed sera from 840 individuals (11-93 years), including sera from reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2-positive (n = 218) and -negative (n = 120) cases, paired sera from SARS-CoV-2 vaccination (n = 29) and infection (n = 11), and paired sera from influenza vaccination (n = 56) and RT-PCR-confirmed influenza infection (n = 158) cases. Last, we analyzed sera collected from 377 individuals who exhibited acute respiratory illness (ARI) in 2020. RESULTS: This 41-plex assay has high sensitivity and specificity in detecting SARS-CoV-2 infections. It differentiated SARS-CoV-2 vaccination (antibody responses only to spike protein) from infection (antibody responses to both spike and nucleoprotein). No cross-reactive antibodies were induced to SARS-CoV-2 from influenza vaccination and infection, and vice versa, suggesting no interaction between SARS-CoV-2 and influenza antibody responses. However, cross-reactive antibodies were detected between spike proteins of SARS-CoV-2 and common human coronaviruses that were removed by serum adsorption. Among 377 individuals who exhibited ARI in 2020, 129 were influenza positive; none had serological evidence of SARS-CoV-2/influenza coinfections. CONCLUSIONS: Multiplex detection of antibody landscapes can provide in-depth analysis of the antibody protective immunity to SARS-CoV-2 in the context of other respiratory viruses, including influenza.

The Pivotal Role of Quantum Dots-Based Biomarkers Integrated with Ultra-Sensitive Probes for Multiplex Detection of Human Viral Infections.

The spread of viral diseases has caused global concern in recent years. Detecting viral infections has become challenging in medical research due to their high infectivity and mutation. A rapid and accurate detection method in biomedical and healthcare segments is essential for the effective treatment of pathogenic viruses and early detection of these viruses. Biosensors are used worldwide to detect viral infections associated with the molecular detection of biomarkers. Thus, detecting viruses based on quantum dots biomarkers is inexpensive and has great potential. To detect the ultrasensitive biomarkers of viral infections, QDs appear to be a promising option as biological probes, while physiological components have been used directly to detect multiple biomarkers simultaneously. The simultaneous measurement of numerous clinical parameters of the same sample volume is possible through multiplex detection of human viral infections, which reduces the time and cost required to record any data point. The purpose of this paper is to review recent studies on the effectiveness of the quantum dot as a detection tool for human pandemic viruses. In this review study, different types of quantum dots and their valuable properties in the structure of biomarkers were investigated. Finally, a vision for recent advances in quantum dot-based biomarkers was presented, whereby they can be integrated into super-sensitive probes for the multiplex detection of human viral infections.

A Rotatable Paper Device Integrating Reverse Transcription Loop-Mediated Isothermal Amplification and a Food Dye for Colorimetric Detection of Infectious Pathogens.

In this study, we developed a rotatable paper device integrating loop-mediated isothermal amplification (RT-LAMP) and a novel naked-eye readout of the RT-LAMP results using a food additive, carmoisine, for infectious pathogen detection. Hydroxyl radicals created from the reaction between CuSO4 and H2O2 were used to decolor carmoisine, which is originally red. The decolorization of carmoisine can be interrupted in the presence of DNA amplicons produced by the RT-LAMP reaction due to how DNA competitively reacts with the hydroxyl radicals to maintain the red color of the solution. In the absence of the target DNA, carmoisine is decolored, owing to its reaction with hydroxyl radicals; thus, positive and negative samples can be easily differentiated based on the color change of the solution. A rotatable paper device was fabricated to integrate the RT-LAMP reaction with carmoisine-based colorimetric detection. The rotatable paper device was successfully used to detect SARS-CoV-2 and SARS-CoV within 70 min using the naked eye. Enterococcus faecium spiked in milk was detected using the rotatable paper device. The detection limits for the SARS-CoV-2 and SARS-CoV targets were both 103 copies/µL. The rotatable paper device provides a portable and low-cost tool for detecting infectious pathogens in a resource-limited environment.

Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses.

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

Multiplex Digital Polymerase Chain Reaction Technology and Its Application

Digital polymerase chain reaction (PCR), as a nucleic acid detection technology with wide application prospect, has become one of the most accurate nucleic acid detection technology at present. Multiplex detection is an important direction for the development of digital PCR technique. With the development of microfluidic technology, multiplex digital PCR technique has become more and more mature. This paper reviewed the research progresses of multiplex digital PCR in recent years, especially summarized the implementation of multiplex digital PCR technique in the past five years, and introduced the application of multiplex digital PCR technique in hot areas such as liquid biopsy, transgenic detection, and SARS-Cov-2 detection. Finally, the issues and challenges faced by multiplex digital PCR technique were discussed and the future direction of the technology was foreseen.

The Burden of Respiratory Viruses and Their Prevalence in Different Geographical Regions of India: 1970-2020.

As per the 2019 report of the National Health Portal of India, 41,996,260 cases and 3,740 deaths from respiratory infections were recorded across India in 2018. India contributes to 18% of the global population, with severe acute respiratory infection (SARI) as one of the prominent causes of mortality in children >5 years of age. Measures in terms of the diagnosis and surveillance of respiratory infections are taken up globally to discover their circulating types, detect outbreaks, and estimate the disease burden. Similarly, the purpose of this review was to determine the prevalence of respiratory infections in various regions of India through published reports. Understanding the pattern and prevalence of various viral entities responsible for infections and outbreaks can help in designing better strategies to combat the problem. The associated pathogens comprise respiratory syncytial virus (RSV), rhinovirus, influenza virus, parainfluenza virus, adenovirus, etc. Identification of these respiratory viruses was not given high priority until now, but the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has sensitized our system to be alert about the burden of existing infections and to have proper checks for emerging ones. Most of the studies reported to date have worked on the influenza virus as a priority. However, the data describing the prevalence of other respiratory viruses with their seasonal pattern have significant epidemiological value. A comprehensive literature search was done to gather data from all geographical regions of India comprising all states of India from 1970 to 2020. The same has been compared with the global scenario and is being presented here.

A fully automated microfluidic PCR-array system for rapid detection of multiple respiratory tract infection pathogens.

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL-1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

3' Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System.

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3' Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.