Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.

Near-Infrared Responsive Ag@Au Nanoplates with Exceptional Stability for Highly Sensitive Colorimetric and Photothermal Dual-Mode Lateral Flow Immunoassay.

Lateral flow immunoassay (LFIA) has played a vital role in point-of-care (POC) testing on account of its simplicity, rapidity, and low cost. However, the low sensitivity and difficulty of quantitation limit its further development. Sensitive markers with new detection modes are being developed to dramatically improve LFIA's performance. Herein, a ligand-complex approach was proposed to uniformly coat a thin layer of Au onto Ag triangular nanoplates (Ag TNPs) without etching the Ag cores, which not only retain the unique optical properties from Ag TNPs but also acquire the surface stability and biocompatibility of gold. The localized surface plasmon resonance absorption of these Ag@Au TNPs could be finely adjusted from visible (550 nm) to the second near-infrared region (NIR-II) (1100 nm), and even longer, by simply adjusting the ratio between edge length and thickness. Utilizing the Ag@Au TNPs as new markers for LFIA, a highly sensitive colorimetric and photothermal dual-mode detection of the SARS-CoV-2 nucleocapsid protein was achieved with a very low background. The Ag@Au TNPs showed an exceedingly high photothermal conversion efficiency of 61.4% (ca. 2 times higher than that of Au nanorods), endowing the LFIA method with a low photothermal detection limit (40 pg/mL), which was 25-fold lower than that of the colorimetric results. The generality of the method was further verified by the sensitive and accurate analysis of cardiac troponin I (cTnI). This method is robust, reproducible, and highly specific and has been successfully applied to SARS-COV-2 detection in 35 clinical samples with satisfactory results, demonstrating its potential for POC applications.

PEI-Mediated Assembly of Fe3O4 onto SiO2-Encapsulated CsPbBr3 for Highly Sensitive Fluorescent Lateral Flow Immunoassay.

The conventional lateral flow immunoassay (LFIA) method using colloidal gold nanoparticles (Au NPs) as labeling agents faces two inherent limitations, including restricted sensitivity and poor quantitative capability, which impede early viral infection detection. Herein, we designed and synthesized CsPbBr3 perovskite quantum dot-based composite nanoparticles, CsPbBr3@SiO2@Fe3O4 (CSF), which integrated fluorescence detection and magnetic enrichment properties into LFIA technology and achieved rapid, sensitive, and convenient quantitative detection of the SARS-CoV-2 virus N protein. In this study, CsPbBr3 served as a high-quantum-yield fluorescent signaling probe, while SiO2 significantly enhanced the stability and biomodifiability of CsPbBr3. Importantly, the SiO2 shell shows relatively low absorption or scattering toward fluorescence, maintaining a quantum yield of up to 74.4% in CsPbBr3@SiO2. Assembly of Fe3O4 nanoparticles mediated by PEI further enhanced the method's sensitivity and reduced matrix interference through magnetic enrichment. Consequently, the method achieved a fluorescent detection range of 1 × 102 to 5 × 106 pg·mL-1 after magnetic enrichment, with a limit of detection (LOD) of 58.8 pg·mL-1, representing a 13.3-fold improvement compared to nonenriched samples (7.58 × 102 pg·mL-1) and a 2-orders-of-magnitude improvement over commercial colloidal gold kits. Furthermore, the method exhibited 80% positive and 100% negative detection rates in clinical samples. This approach holds promise for on-site diagnosis, home-based quantitative tests, and disease procession evaluation.

Do renewable energy, environmental regulations and green innovation matter for China's zero carbon transition: Evidence from green total factor productivity.

Rapid developments in environmental infrastructure have contributed to significant improvements in green total factor productivity, but further investigation is required to provide a detailed assessment to understand the policy mechanisms involved. This paper analyzes environmental progress in China through MMQR, CCEMG, and AMG as empirical strategies for 30 provinces in China. Our empirical results reveal that energy optimization through renewable energy is the most effective channel to improve green total factor productivity, though it is not the only available option. Since environmental regulations, infrastructure development, and green technology innovation also directly impact energy efficiency, adopting these within policy channels will positively impact environmental sustainability. Our empirical approach helps suggest novel environmental policy suggestions. In particular, policymakers must introduce structural changes within energy developments to foster renewable energy. Furthermore, China must increase environmental spending to upgrade its energy infrastructure further and solve ecological issues. These insights offer valuable policy guidance for decision-makers in China and globally, aiming to foster economic and environmental sustainability and achieve zero-carbon transition goals.

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

One-Year Sustained Cellular and Humoral Immunities in Coronavirus Disease 2019 (COVID-19) Convalescents.

BACKGROUND: The longitudinal antigen-specific immunity in COVID-19 convalescents is crucial for long-term protection upon individual re-exposure to SARS-CoV-2, and even more pivotal for ultimately achieving population-level immunity. We conducted this cohort study to better understand the features of immune memory in individuals with different disease severities at 1 year post-disease onset. METHODS: We conducted a systematic antigen-specific immune evaluation in 101 COVID-19 convalescents, who had asymptomatic, mild, moderate, or severe disease, through 2 visits at months 6 and 12 after disease onset. The SARS-CoV-2-specific antibodies, comprising neutralizing antibody (NAb), immunoglobulin (Ig) G, and IgM, were assessed by mutually corroborated assays (ie, neutralization, enzyme-linked immunosorbent assay [ELISA], and microparticle chemiluminescence immunoassay [MCLIA]). Meanwhile, T-cell memory against SARS-CoV-2 spike, membrane, and nucleocapsid proteins was tested through enzyme-linked immunospot assay (ELISpot), intracellular cytokine staining, and tetramer staining-based flow cytometry, respectively. RESULTS: SARS-CoV-2-specific IgG antibodies, and NAb, can persist among >95% of COVID-19 convalescents from 6 to 12 months after disease onset. At least 19/71 (26%) of COVID-19 convalescents (double positive in ELISA and MCLIA) had detectable circulating IgM antibody against SARS-CoV-2 at 12 months post-disease onset. Notably, numbers of convalescents with positive SARS-CoV-2-specific T-cell responses (≥1 of the SARS-CoV-2 antigen S1, S2, M, and N proteins) were 71/76 (93%) and 67/73 (92%) at 6 and 12 months, respectively. Furthermore, both antibody and T-cell memory levels in the convalescents were positively associated with disease severity. CONCLUSIONS: SARS-CoV-2-specific cellular and humoral immunities are durable at least until 1 year after disease onset.

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay.

CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.

Ag Nanoparticles with Ultrathin Au Shell-Based Lateral Flow Immunoassay for Colorimetric and SERS Dual-Mode Detection of SARS-CoV-2 IgG.

Immunoglobulin detection is essential for diagnosing progression of SARS-CoV-2 infection, for which SARS-CoV-2 IgG is one of the most important indexes. In this paper, Ag nanoparticles with ultrathin Au shells (∼2 nm) embedded with 4-mercaptobenzoic acid (MBA) (AgMBA@Au) were manufactured via a ligand-assisted epitaxial growth method and integrated into lateral flow immunoassay (LFIA) for colorimetric and SERS dual-mode detection of SARS-CoV-2 IgG. AgMBA@Au possessed not only the surface chemistry advantages of Au but also the superior optical characteristics of Ag. Moreover, the nanogap between the Ag core and the Au shell also greatly enhanced the Raman signal. After being modified with anti-human antibodies, AgMBA@Au recognized and combined with SARS-CoV-2 IgG, which was captured by the SARS-CoV-2 spike protein on the T line. Qualitative analysis was achieved by visually observing the color of the T line, and quantitative analysis was conducted by measuring the SERS signal with a sensitivity four orders of magnitude higher (detection limit: 0.22 pg/mL). The intra-assay and inter-assay variation coefficients were 7.7 and 10.3%, respectively, and other proteins at concentrations of 10 to 20 times higher than those of SARS-CoV-2 IgG could hardly produce distinguishable signals, confirming good reproducibility and specificity. Finally, this method was used to detect 107 clinical serum samples. The results agreed well with those obtained from enzyme-linked immunosorbent assay kits and were significantly better than those of the colloidal gold test strips. Therefore, this dual-mode LFIA has great potential in clinical practical applications and can be used to screen and trace the early immune response of SARS-CoV-2.

A CRISPR/Cas9 eraser strategy for contamination-free PCR end-point detection.

Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.

Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay.

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 µL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.

Early Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies as a Serologic Marker of Infection in Patients With Coronavirus Disease 2019.

BACKGROUND: Thousands of medical staff have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with hundreds of deaths reported. Such loss could be prevented if there were a serologic assay for SARS-CoV-2-specific antibodies for serological surveillance of its infection at the early stage of disease. METHODS: Using Chinese hamster ovarian (CHO) cell-expressed full-length SARS-CoV-2 S1 protein as capturing antigen, a coronavirus disease 2019 (COVID-19)/SARS-CoV-2 S1 serology enzyme-linked immunosorbent assay (ELISA) kit was developed and validated with negative samples collected prior to the outbreak or during the outbreak and positive samples from patients confirmed with COVID-19. RESULTS: The specificity of the ELISA kit was 97.5%, as examined against total 412 normal human samples. The sensitivity was 97.1% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. The assay was able to detect SARS-CoV-2 antibody on day 1 after the onset of COVID-19 disease. The average antibody levels increased during hospitalization and 14 days after discharge. SARS-CoV-2 antibodies were detected in 28 of 276 asymptomatic medical staff and 1 of 5 nucleic acid test-negative "close contacts" of COVID-19 patients. CONCLUSIONS: With the assays developed here, we can screen medical staff, incoming patients, passengers, and people who are in close contact with the confirmed patients to identify the "innocent viral spreaders," protect the medical staff, and stop further spread of the virus.

One-Step Monitoring of Multiple Enterovirus 71 Infection-Related MicroRNAs Using Core-Satellite Structure of Magnetic Nanobeads and Multicolor Quantum Dots.

The accurate and rapid monitoring of the expression levels of enterovirus 71 (EV71)-related microRNAs (miRNAs) can contribute to diagnosis of hand, foot, and mouth disease (HFMD) at the early stage. However, there is currently a lack of convenient methods for simultaneous monitoring of multiplex miRNAs in one step. Herein a one-step method for the simultaneous monitoring of multiple EV71 infection-related miRNAs is developed based on core-satellite structure assembled with magnetic nanobeads and quantum dots (MNs-ssDNA-QDs). In the presence of target miRNAs, duplex-specific nuclease (DSN)-assisted target recycling can be triggered, resulting in the release of QDs and recycling of target miRNAs. Then the simultaneous quantification can be easily realized by recording the corresponding amplified fluorescence signal of QDs in the suspension. With this method, simultaneous detection of hsa-miRNA-296-5p and hsa-miRNA-16-5p, potential biomarkers of EV71 infection, can be easily achieved with femtomolar sensitivity and single-base mismatch specificity. Moreover, the method is successfully used for monitoring of the expression level of miRNAs in EV71-infected cells at different time points, demonstrating the potential for diagnostic applications. With the merits of one-step operation and single-nucleotide mismatch discrimination, this work opens a new avenue for multiplex miRNAs detection. As different nucleotide sequences and multicolor QDs can be employed, this work is expected to offer great potential for the development of high throughput diagnosis.

Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.

Rapid screening and quantitative detection of Salmonella using a quantum dot nanobead-based biosensor.

The continuing hurdle of developing foodborne pathogen detection techniques is that compromises must be made among simplicity, portability, speed, sensitivity, and quantitation. Herein, we fabricated quantum dot nanobeads (QDNS) by a layer-by-layer assembly of quantum dots on the surface of polymer nanospheres. QDNS exhibited higher fluorescence intensity than the quantum dots at the same particle number. Based on the quantum dot nanobeads as the signal reporter, a quantitative lateral flow immunoassay was demonstrated for Salmonella typhimurium detection with improved sensitivity, specificity and accuracy. A visual detection limit of 5 × 103 CFU mL-1Salmonella typhimurium within 10 min has been proved and demonstrated. Additionally, higher concentrations of non-Salmonella typhimurium bacteria have negligible effects on the detection of Salmonella typhimurium. The results of 50 single blind tests by 10 testers suggested that the assay exhibited 100% accuracy. The results illustrate that the assay provides a balance among simplicity, speed, sensitivity and accuracy, and it can be a favorable alternative for Salmonella typhimurium screening in various samples.

Neisseria meningitidis carriage and risk factors among teenagers in Suizhou city in China.

Teenagers are important carriers of Neisseria meningitidis, which is a leading cause of invasive meningococcal disease. In China, the carriage rate and risk factors among teenagers are unclear. The present study presents a retrospective analysis of epidemiological data for N. meningitidis carriage from 2013 to 2017 in Suizhou city, China. The carriage rates were 3.26%, 2.22%, 3.33%, 3.53% and 9.88% for 2013, 2014, 2015, 2016 and 2017, respectively. From 2014 to 2017, the carriage rate in the 15- to 19-year-old age group (teenagers) was the highest and significantly higher than that in remain age groups. Subsequently, a larger scale survey (December 2017) for carriage rate and relative risk factors (population density, time spent in the classroom, gender and antibiotics use) were investigated on the teenagers (15- to 19-year-old age) at the same school. The carriage rate was still high at 33.48% (223/663) and varied greatly from 6.56% to 52.94% in a different class. Population density of the classroom was found to be a significant risk factor for carriage, and 1.4 persons/m2 is recommended as the maximum classroom density. Further, higher male gender ratio and more time spent in the classroom were also significantly associated with higher carriage. Finally, antibiotic use was associated with a significantly lower carriage rate. All the results imply that attention should be paid to the teenagers and various measures can be taken to reduce the N. meningitidis carriage, to prevent and control the outbreak of IMD.

Colorimetric-Fluorescent-Magnetic Nanosphere-Based Multimodal Assay Platform for Salmonella Detection.

Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multisignal readout lateral flow immunoassay for Salmonella typhimurium ( S. typhi) detection. The assay employs colorimetric-fluorescent-magnetic nanospheres (CFMNs) as labels, which possess multifunctional target separation and enrichment, multisignal readout, and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labeled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88 × 104 CFU/mL S. typhi, and 3.75 × 103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2-4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88 × 104 to 1.88 × 107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum, and whole blood) indicated its potential application in real samples.

Multifunctional Cellular Beacons with in Situ Synthesized Quantum Dots Make Pathogen Detectable with the Naked Eye.

The rapid and sensitive detection of pathogens is extremely crucial for timely clinical diagnosis and diseases control. Here, by employing cellular beacons with in situ synthesized QDs created from Staphylococcus aureus ( S. aureus), we efficiently fabricated an antibody (Ab) and acetylcholinesterase (AChE)-functionalized nanobioprobe, i.e., multifunctional cellular beacons (MCBs), avoiding complicated modification. Coupled with magnetic separation, a novel method for pathogen detection with the naked eye is established. With this method, enterovirus 71 (EV71) can be detected by the naked eye through the aggregation of gold nanoparticles that is triggered by the product of AChE catalyzed acetylthiocholine, with a detection limit of 0.5 ng/mL. Moreover, due to the MCBs have high luminance with perfect uniformity, the detection can also be realized by counting the number of MCBs, with a detection limit of 1 ng/mL. The method is validated with human throat swabs, resulting in a complete consistence with reverse transcription-polymerase chain reaction results. This study reports the first cellular beacons-based method for pathogen detection by the naked eye and broadens the applicability of cell self-synthesized nanoparticles-based immunoassays. Moreover, the MCBs-based method will provide a powerful tool for clinical detection.

Dual-Signal Readout Nanospheres for Rapid Point-of-Care Detection of Ebola Virus Glycoprotein.

Rapid detection of highly contagious pathogens is the key to increasing the probability of survival and reducing infection rates. We developed a sensitive and quantitative lateral flow assay for detection of Ebola virus (EBOV) glycoprotein with a novel multifunctional nanosphere (RNs@Au) as a reporter. Each RNs@Au contains hundreds of quantum dots and dozens of Au nanoparticles and can achieve enhanced dual-signal readout (fluorescence signal for quantitative detection and colorimetric signal for visual detection). Antibody (Ab) and streptavidin (SA) were simultaneously modified onto the RNs@Au to label the target and act as signal enhancer. After the target was labeled by the Ab-RNs@Au-SA and captured on the test line, biotin-modified RNs@Au was used to amplify the dual signal by the reaction of SA with biotin. The assay enables naked-eye detection of 2 ng/mL glycoprotein within 20 min, and the quantitative detection limit is 0.18 ng/mL. Additionally, the assay has been successfully tested in field work for detecting EBOV in spiked urine, plasma, and tap water samples and is thus a promising candidate for early diagnosis of suspect infections in EBOV-stricken areas.

Simultaneous Point-of-Care Detection of Enterovirus 71 and Coxsackievirus B3.

Human enterovirus 71 (EV71) is one of the pathogens that causes hand, foot, and mouth disease (HFMD), which generally leads to neurological diseases and fatal complications among children. Since the early clinical symptoms from EV71 infection are very similar to those from Coxsackievirus B3 (CVB3) infection, a robust and sensitive detection method that can be used to distinguish EV71 and CVB3 is urgently needed for prompting medical treatment of related diseases. Herein, based on immunomagnetic nanobeads and fluorescent semiconductor CdSe quantum dots (QDs), a method for simultaneous point-of-care detection of EV71 and CVB3 is proposed. The synchronous detection of EV71 and CVB3 virions was achieved within 45 min with high specificity and repeatability. The limits of detection are 858 copies/500 µL for EV71 and 809 copies/500 µL for CVB3.This proposed method was further validated with 20 human throat swab samples obtained from EV71 or CVB3 positive cases, with results 93.3% consistent with those by the real-time PCR method, demonstrating the potential of this method for clinical quantification of EV71 and CVB3. The method may also facilitate the prevention and treatment of the diseases.

Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus.

BACKGROUND: Respiratory Syncytial Virus (RSV) is an important human respiratory pathogen, particularly of infants and older adults, and despite several decades of research and development, no licensed vaccine is available. Studies have confirmed that enhancement of RSV disease does not occur after inoculation with RSV live-attenuated vaccine candidates, making such vaccines preferable. In this paper, reverse genetics was used to construct two recombinant viruses, a recombinant Long strain (rLong) and rLong-∆G-EGFP; rLong-∆G-EGFP is a recombinant mutant in which G was replaced with the EGFP gene, based on the Long strain of RSV. RESULTS: Both rLong and rLong-∆G-EGFP were constructed successfully and recovered in Hep-2 cells, and autofluorescence was observed in rLong-∆G-EGFP-infected cells during consecutive passages. Titers of rLong and rLong-∆G-EGFP were ~100-fold lower than the parental strain. Although virulence was attenuated, high titers of neutralizing antibodies were induced in BALB/c mice after being inoculated with recombinant viruses in a three-dose schedule. Unexpectedly, the neutralizing antibody titer in rLong-∆G-EGFP-immunized recipients did not decline significantly compared with the rLong strain. Protective efficacy of recombinant viruses in lung tissue was up to 100%, and the serum neutralizing antibody levels could stabilize at 21 days with no significant fall post-challenge. Enzyme-linked immunospot (ELISPOT) assays showed that both recombinant viruses were capable of inducing CD8+ T cell immune responses, which are crucial for virus clearance, and that rLong stimulated a higher level of IFN-γ production by comparison. In terms of inducing a balanced immune response, rLong-∆G-EGFP elicited slightly higher levels of IgG2a antibodies and lower levels of IgG1/IgG2a than the rLong virus. CONCLUSIONS: This study suggested that immunization with rLong and rLong-∆G-EGFP were immunogenic and protected against RSV infection in the lower respiratory tract of BALB/c mice better than in the nose. Because of a relative low IgG1/IgG2a ratio, rLong-∆G-EGFP was more inclined to make CD4+ T cells, shifting toward a Th1-type response, indicating that the generation of a more balanced Th1/Th2 response was desirable. This explorative study on the recombinant Long viruses also contributed to obtaining more RSV attenuated candidates by a reverse genetics approach.