Infection with the HPV (human papillomavirus) has been linked to an increase in cervical cancer incidence in recent years. Since HPV16 and HPV18 infections account for over half of all cervical cancer cases, testing for these viruses is crucial to preventing cervical cancer. In this study, we report a CRISPR/Cas12a-mediated biosensor for BioLayer Interferometry (BLI) that enables rapid and specific detection of HPV16 and HPV18. The addition signal of BLI traditional nucleic acid detection was converted into a subtractive signal, which effectively reduces false positives and improves reaction specificity. Additionally, this work designed a probe-gold nanoparticle conjugate to further enhance detection sensitivity. This method detects HPV16 and HPV18 as low as 460 fM and 500fM within 40 min, without amplification, avoiding aerosol contamination, and with high specificity. This method provides a feasible solution for the rapid, sensitive, and qualitative detection of HPV16 and HPV18.

Against the backdrop of iterative upgrades in pet pathogen detection technology, rapid on-site testing (POCT) has become the core technology for on-site identification of deadly pet diseases. Based on the research and development of new materials, intelligent sensors with high sensitivity, fast response, and high design flexibility have demonstrated strong application value and have become an important development direction for the next-generation technology system in the field of pet pathogen detection. Herein, we report two advanced intelligent material-integrated biosensing platforms: a DNA hydrogel-encapsulated glucose amylase-based assay (RC-HGPGA) and a magnetic nanoparticles (MNPs)-based system where single-stranded DNA (ssDNA) serves as a molecular bridge to conjugate MNPs with invertase (RC-MBI). Both systems operate via a cascade reaction: recombinase polymerase amplification (RPA) of target nucleic acids first activates Cas12a nuclease, which then exerts trans-cleavage activity toward the biosensing elements. Subsequent enzymatic hydrolysis generates glucose, whose concentration is quantifiable using a commercial personal glucose meter (PGM). All experimental procedures were conducted at a constant temperature of 37 °C, eliminating the need for complex thermal cycling equipment. Our findings demonstrate that the RC-HGPGA and RC-MBI platforms achieve ultra-sensitive detection of feline panleukopenia virus (FPV) and canine distemper virus (CDV)-two clinically significant pet viruses-with limits of detection (LODs) as low as 10° copies/μL and 101 copies/μL, respectively, within a rapid time of 35 min. Both systems exhibit high sensitivity, excellent specificity, broad adaptability, and user-friendliness, thereby showing great potential for on-site detection of pet viruses.

Methicillin-resistant Staphylococcus aureus (MRSA) has become a threat to global public health. It is one of the important pathogens of nosocomial infections and community-acquired infections. The development of highly sensitive detection methods is therefore critical for effective MRSA infection prevention. We present an innovative Ethidium monoazide bromide (EMA)-coupled CRISPR Cas12a-Graphene Field-Effect Transistor (GFET) system (ECGS) for rapid, sensitive, and specific detection of viable MRSA. This method is based on the selective labeling of dead bacterial DNA by EMA, which enables the identification of live bacteria. At the same time, the high specificity of CRISPR single-base recognition and the sensitivity of GFET make it possible to detect the target genome as low as 0.5 aM in 30 min. In this study, we targeted the clfA gene for Staphylococcus aureus (SA) identification and the mecA gene for the mark of methicillin resistance. The ECGS platform establishes a new paradigm for viable pathogen detection with significant implications for food safety and public health.

Involvement of the intestinal autonomic nerves in Guillain-Barré syndrome (GBS) can lead to paralytic ileus, a condition commonly observed in severe cases during later stages of the disease. Cases with paralytic ileus as a presenting symptom are very rare. We report a case of a 35-year-old male patient who was admitted to the hospital with acute abdominal pain persisting for 12 hours. Abdominal CT suggested small bowel obstruction, for which routine conventional pharmacological treatment were ineffective. Subsequently, the patient presented with multiple sets of cranial nerve paralysis, bilateral symmetrical delayed paralysis, distal limb numbness, respiratory failure, urinary retention, shock, and electrophysiology, suggesting axonal-type multifocal peripheral nerve damage. Notably, blood antiganglioside tests showed IgG positivity for anti-sulfatide antibodies, anti-GD1a antibodies, and anti-GT1a antibodies. The patient was administered plasma exchange combined with intravenous immunoglobulin, and symptoms gradually improved. The patient resumed independent ambulation within two months and returned to normal status at one year, with no recurrence of symptoms. Given that paralytic ileus can precede other neurological abnormalities in patients with GBS, early detection and individualized treatment are critical to reduce the risk of death and promote recovery. Here, we demonstrate that intensive immunotherapy is a viable therapeutic approach that can be clinically adopted for such conditions.

Duck viral hepatitis is very common in China, causing significant impact and economic losses to the duck farming industry. Currently, DHAV-3 has become the main factor causing duck viral hepatitis in China. However, the existing DHAV-3 vaccines cannot completely rule out the potential risk of the vaccine strain becoming more virulent. Due to the high similarity in genomic sequences between wild strains and vaccine strains (with only a few base differences), traditional detection methods struggle to accurately differentiate them, severely interfering with disease control decisions. Therefore, simultaneously detecting both the virulent strain and the attenuated strain of DHAV-3 is crucial for evaluating vaccine efficacy, monitoring virus mutations, and optimizing control strategies. This study, using the DHAV-3 SD70 attenuated strain as an example, developed a highly sensitive and rapid detection method to identify and distinguish between the DHAV-3 virulent and SD70 attenuated strains, providing a new strategy for identifying both strains. Currently, there are no literature reports on the detection methods for the two strains. Therefore, we propose a single-base recognition system strategy based on RPA-CRISPR. DHAV-3 virulent and attenuated strains were specifically identified by this method based on only a few different base sequences. This method can detect two target genes as low as 10° copy/μL within 35 min. In addition, when this method was used for samples analysis, the results of this method, sequencing results, and the results provided by the company were compared and found to be consistent. This method has the advantages of fast speed, simple operation, high specificity and sensitivity, which can be used for the detection of DHAV-3 virulence strain and SD70 attenuated strain, and lays a technical foundation for disease control, vaccine evaluation and mutation monitoring.

Here, we report a novel, vertical flow, and regenerable CRISPR-Cas12a biosensor based on surface plasmon resonance (SPR) for nucleic acid detection of viruses. The CRISPR-SPR biosensor is designed to be regenerative and amplification-free, based on nucleic acid hybridization and special sequences to capture and release reporter DNA. Additionally, gold nanoparticles (AuNPs) enhance the response signal for further improving the sensitivity of nucleic acid detection. After experimental optimization, the limit of detection (LOD) of the CRISPR-SPR biosensor was 3.75 pM for cytomegalovirus (CMV) templates. The potential of the sensor in clinical sample detection was analyzed using t-test, which revealed a significant difference between the positive and negative CMV samples (P < 0.001). The vertical multi-channel and regenerative design of the CRISPR-SPR sensor could reduce non-specific adsorption as well as usage costs. The coefficient of variation (CV) was 2.76 % for the SPR response signal of the reporter gene after 50 repeated experiments, significantly enhancing the reproducibility and consistency of the experiments. The regenerative biosensor would offer good economic benefits and shows great potential in the detection of nucleic acid-related diseases.

This study is concerned with the development of a long non-coding RNA (lncRNA) BCRT1 bio-platform based on tetrahydroxyborate-bismuth vandate ([B(OH)4]--BiVO4) for early diagnosis of cervical cancer (CC). A biosensor based on ([B(OH)4]--BiVO4 was constructed towards CC exosomal lncRNA biomarker. Formation of [B(OH)4]- ligand passivated BiVO4 helps to eliminate surface defects, reducing charge recombination. [B(OH)4]--BiVO4 serves as a base material with excellent photoelectric properties, showing a signal response up to 0.89 mA∙cm-2. This study focused on designing a capture probe for exosomal lncRNA BCRT1 in order to effectively detect early warning signs of the CC. The probe achieved a low detection limit of 5.53 fmol∙L-1 within a range of 0.01-10000 pmol∙L-1, demonstrating good stability, reproducibility and selectivity. This research offers a promising method for the early diagnosis of CC.

Cervical cancer is the second leading cause of cancer death among women in developing countries. The most common screening methods for cervical cancer are cytology and HPV DNA detection, but their sensitivity and specificity are limited. Early detection of cervical cancer biomarkers can improve screening accuracy and help clinicians make timely diagnoses, predict treatment responses, and monitor disease progression. Here, we report a dual RPA-CRISPR protein detection system (DRC-PDS) for ultrasensitive quantitative detection of cervical cancer tumor marker proteins SCCA and CEA. This method adapts CRISPR-based nucleic acid detection assays for detecting proteins that utilize Cas12a and Cas13a for dual-target detection in a single tube, enabling the detection of marker proteins as low as 10 aM. At the same time, this method has high specificity. The entire reaction process can be completed in a single step with a handheld instrument without opening the lid, avoiding aerosol contamination during the reaction. Combined with a handheld instrument, the detection system has great potential for use in the community and at the point of temporary care.

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are two common herpes viruses that cause infections with overlapping clinical symptoms, making clinical diagnosis challenging. CMV infection has a high prevalence, affecting over 50 % of the global population, and poses serious risks to immunocompromised individuals and newborns. EBV, with an infection rate exceeding 90 % worldwide, is primarily associated with infectious mononucleosis and has been implicated in the development of several malignancies. Given their distinct clinical implications and the need for timely and accurate diagnosis, the development of a reliable detection method is essential. In this study, we combine the Cas12a system with a T7 transcription-mediated Cas13a system to establish a single-tube, dual RPA CRISPR-Cas detection system (DRCDS) to identify CMV and EBV at the same time. This method demonstrated high sensitivity, capable of detecting as low as 1 copy/mu L targets within 35 min, along with excellent specificity. This approach provides a rapid, accurate, and efficient diagnostic solution for the clinical differentiation of CMV and EBV, offering a valuable alternative for clinical applications.

Bacteria and fungi are abundant and ubiquitous in bioaerosols in hospital environments. Understanding the distribution and diversity of microbial communities within bioaerosols is critical for mitigating their detrimental effects. Our knowledge on the composition of bacteria or fungi in bioaerosols is limited, especially the potential pathogens present in fine particulate matter (PM2.5) from specialized hospitals. Thirty p.m.(2.5) filter samples were collected from five hospitals (i.e., oral, dermatology, chest, eye, and general hospitals) in Shandong Province, East China. The diversity of bacteria and fungi was analyzed at the species level using single-molecule real-time sequencing of the 16 S and internal transcribed spacer 1 (ITS) ribosomal genes, respectively. Significant differences were detected across sampling sites in terms of microbial diversity and community composition in PM2.5 as well as pollution concentrations. The range of PM2.5 concentrations observed in hospital halls was higher, ranging from 39.0 to 46.2 mu g/m(3), compared to the wards where the concentrations ranged from 10.7 to 25.2 mu g/m(3). Furthermore, microbial variations in PM2.5 bioaerosols were associated with hospital type. The most dominant pathogens identified were Vibrio metschnikovii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Fusarium pseudensiforme, and Aspergillus ruber. Among these, A. ruber was identified as an opportunistic fungus in a hospital setting for the first time. Nine potentially novel strains of F. pseudensiforme, showing 84.5%-92.0% ITS sequence similarity to known Fusarium isolates, were identified in PM2.5 samples from all hospitals (excluding an eye hospital). This study highlights the importance of hospital environments in shaping microbial aerosol communities. To the best of our knowledge, this is the first study to provide insights into the bacterial and fungal biodiversity of PM2.5 in specialized hospitals, enriching research in healthcare environmental microbiology and carrying significant public health implications.

Bacterial and fungal aerosol pollution is widespread in indoor school environments, and poses potential health risks to students and staff. Understanding the distribution and diversity of microbial communities within aerosols is crucial to mitigate their adverse effects. Existing knowledge regarding the composition of bacterial and fungal aerosols, particularly the presence of potential pathogenic microorganisms in fine particulate matter (PM2.5) from nursery schools to universities, is limited. To bridge this knowledge gap, in the present study, we collected PM2.5 samples from five types of schools (i.e., nursery schools, primary schools, junior schools, and high schools and universities) in China. We used advanced single-molecule real-time sequencing to analyze the species-level diversity of bacterial and fungal components in PM2.5 samples based on 16S and ITS ribosomal genes, respectively. We found significant differences in microbial diversity and community composition among the samples obtained from different educational institutions and pollution levels. In particularly, junior schools exhibited higher PM2.5 concentrations (62.2-86.6 μg/m3) than other schools (14.4-48.4 μg/m3). Moreover, microbial variations in PM2.5 samples were associated with institution type. Notably, the prevailing pathogenic microorganisms included Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, and Schizophyllum commune, all of which were identified as Class II Pathogenic Microorganisms in school settings. Four potentially novel strains of S. commune were identified in PM2.5 samples collected from the university; the four strains showed 92.4 %-94.1 % ITS sequence similarity to known Schizophyllum isolates. To the best of our knowledge, this is the first study to explore bacterial and fungal diversity within PM2.5 samples from nursery schools to universities. Overall, these findings contribute to the existing knowledge of school environmental microbiology to ensure the health and safety of students and staff and impacting public health.

We developed a system that integrates RPA-CRISPR Cas12a with microfluidic chip, allowing for the rapid, highly specific, and sensitive detection and differentiation of SARS-CoV-2, H1N1, and H3N2.

The incidence of cervical cancer caused by human papillomavirus (HPV) infection has increased in recent years. More than half of all cervical cancer cases are due to HPV16 and HPV18 infection, so HPV16 and HPV18 testing is essential to prevent cervical cancer. HPV testing is mainly carried out in hospitals, but it is subject to time and specialized medical facilities. On the other hand, home self-testing using simple diagnostics would present an attractive alternative due to privacy and flexibility with regard to time and place, provided sufficient sensitivity and specificity can be achieved. In this work, a dual lateral flow assay based on RPA-CRISPR-Cas12a/13a (named RC-LFA) for HPV detection was described. Taking advantage of the cleavage specificity of Cas12a and Cas13a, a CRISPR-Cas12a/Cas13a system was designed to detect HPV16 and HPV18. The lateral flow strip with two test lines was designed to suit the CRISPR-Cas12a/Cas13 system. RC-LFA achieves rapid and simultaneous detection of HPV16 and HPV18 with high specificity and sensitivity (10 copies/μL) in about 40 min from the extraction of nucleic acid to an instrument-free readout. RC-LFA is user-friendly and instrument-free, making it a promising method for HPV self-tests at home.

The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On -site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one -pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 10 0 copy/ mu L, while DRC-LFA achieved limit of 10 1 copies/ mu L within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on -site virus detection.

Microbial aerosol contamination is a common problem in poultry farms, posing potential health risks to poultry and their caretakers. Exploring the distribution and diversity of the microbial community in poultry farm aerosols is crucial for effective mitigation strategies. The composition of bacterial and fungal aerosols is poorly understood, particularly the prevalence of potential pathogenic microorganisms in fine particulate matter (PM2.5) in various types of poultry houses. In this study, 27 PM2.5 samples were collected from 5 chicken houses and 4 duck houses in Shandong Province, China. Species-level diversity of bacterial and fungal components in PM2.5 samples was determined using advanced single-molecule real-time sequencing (SMRT) technology, based on the 16S and internal transcribed spacer 1 (ITS) ribosomal genes. Microbial diversity and community composition varied significantly across the different poultry house. Notably, duck houses had higher concentrations (p < 0.01) of PM2.5 (92.8-143.1 mu g/m(3)) than chicken houses (42.0-56.4 mu g/m(3)). Furthermore, microbial variation in PM2.5 samples was associated with the type of poultry facility. The predominant pathogenic microorganisms included Aspergillus sydowii, Penicillium sp., Aspergillus insolitus, Cladosporium sp., Aspergillus sp., Aspergillus pseudoglaucus, Cladosporium sp. C4092-2-PD1, and Colletotrichum sp., all of which were classified as second category of pathogens. Aspergillus sydowii and Penicillium sp. were the dominant species in chicken houses, while Cladosporium sp., Aspergillus sp., and Aspergillus pseudoglaucus were the dominant species identified in duck houses. To our knowledge, this study is the first to investigate bacterial and fungal diversity in PM2.5 samples collected from various types of poultry houses. These findings advance our understanding of poultry environmental microbiology and have important implications for safeguarding the health of both poultry and their caretakers.

Intelectin is a lectin with the capacity to recognize and bind to carbohydrates. In this study, we successfully cloned cITLN3 from common carp, which consists of a signal peptide domain, a FReD domain, and an intelectin domain. The expression levels of cITLN3 were detected in various organs of common carp, including the liver, head kidney, spleen, foregut, midgut, and hindgut, with the highest expression observed in the liver. Following infection with Staphylococcus aureus (S. aureus) or Aeromonas hydrophila (A. hydrophila), the expression level of cITLN3 was significantly upregulated in all organs of common carp. Subsequently, we expressed and purified the recombinant cITLN3 protein using an E. coli expression system. The cITLN3 could aggregate both gram-positive and gram-negative bacteria in the presence of Ca2+, with a stronger affinity for gram-positive bacteria. Moreover, our study demonstrated that cITLN3 displayed a higher binding affinity towards PGN compared to LPS. Furthermore, we observed that cITLN3 had the potential to inhibit bacterial proliferation in common carp and safeguard intestinal integrity during bacterial stimulation. And the results also indicated that cITLN3 might played a role in the Toll-like receptors (TLRs) signaling pathway activation.

Foot-and-mouth disease virus (FMDV) has developed various strategies to antagonize the host innate immunity. FMDV Lpro and 3Cpro interfere with type I IFNs through different mechanisms. The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γ signaling transduction. Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown. In this study, it was shown that FMDV replication was resistant to IFN-γ treatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes (ISGs). We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation. 3Cpro expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences (GAS) promoter activity, without affecting the protein level, tyrosine phosphorylation, and homodimerization of STAT1. Finally, we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities. Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.