Search Results (23)

Background: Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease characterized by reproductive failure in sows and severe respiratory disorders across all swine ages, causing significant economic losses. In China, the PRRSV epidemiological landscape is complex, with the coexistence of multiple lineages and frequent recombination. The major circulating strains include sublineages 1.8 (NADC30-like PRRSV) and 1.5 (NADC34-like PRRSV), along with lineages 8 (HP-like PRRSV) and 5 (VR2332-like PRRSV), highlighting the urgent need for rapid detection and lineage differentiation. Methods: A quadruplex RT-qPCR assay was developed targeting lineage-specific deletions in the NSP2 gene to simultaneously detect PRRSV-2 and differentiate NADC30-like PRRSV, HP-like PRRSV, and NADC34-like PRRSV strains. The assay was optimized with respect to reaction conditions, including annealing temperature, primers, and probe concentrations. The method’s performance was evaluated in terms of specificity, sensitivity, repeatability, stability, limit of detection (LOD), and consistency with sequencing results. Results: The assay demonstrated high sensitivity (LOD of 3 copies/μL), high specificity, and good repeatability (coefficient of variation < 1.5%). Field application using 938 samples from Guangxi A and B farms revealed NADC30-like PRRSV wild-type strains at positivity rates of 13.44% and 3.53%, respectively. Positive samples selected for sequencing were further confirmed using ORF5-based phylogenetic analysis and NSP2 deletion pattern comparison, which aligned with RT-qPCR detection results. Field application primarily detected NADC30-like PRRSV, while further validation is still needed for HP-like and NADC34-like strains. The developed quadruplex RT-qPCR assay enables rapid and simultaneous detection of PRRSV-2 and differentiation of three major lineages, providing a sensitive, specific, and reliable tool for distinguishing vaccine-derived from circulating strains and supporting targeted disease surveillance and control in swine farms. Full article

CRISPR/Cas-based diagnostics offer unparalleled specificity, but their reliance on fluorescently labeled probes and complex nucleic acid extraction limits field applicability. To tackle this problem, we have developed a label-free, equipment-free platform integrating FTA card-based extraction, CRISPR/Cas12a, and pre-folded G-quadruplex (G4)–Thioflavin T (ThT) signal reporter. This system eliminates costly fluorescent labeling by leveraging G4-ThT structural binding for visible fluorescence output, while FTA cards streamline nucleic acid isolation without centrifugation. Achieving a limit of detection (LOD) to 10¹ CFU/mL for Escherichia coli O157:H7 in spiked food samples, the platform demonstrated 100% concordance with qPCR and standard fluorescent probe-based CRISPR/Cas12a system. Its simplicity, minimal equipment (portable heating/imaging), and cost-effectiveness make it a revolutionary tool for detecting foodborne pathogens in resource-limited environments. Full article

Porcine sapelovirus (PSV), porcine kobuvirus (PKV), porcine teschovirus (PTV), and porcine enterovirus G (EV-G) are all important viruses in the swine industry. These viruses play important roles in the establishment of similar clinical signs of diseases in pigs, including diarrhea, encephalitis, and reproductive and respiratory disorders. The early accurate detection of these viruses is crucial for dealing with these diseases. In order for the differential detection of these four viruses, specific primers and TaqMan probes were designed for the conserved regions in the 5′ untranslated region (UTR) of these four viruses, and one-step quadruplex reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PSV, PKV, PTV, and EV-G was developed. The results showed that this assay had the advantages of high sensitivity, strong specificity, excellent repeatability, and simple operation. Probit regression analysis showed that the assay obtained low limits of detection (LODs) for PSV, PKV, PTV, and EV-G, with 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively. The assay showed a strong specificity of detecting only PSV, PKV, PTV, and EV-G, and had no cross-reactivity with other control viruses. The assay exhibited excellent repeatability of the intra-assay coefficient of variation (CV) of 0.28–1.58% and the inter-assay CV of 0.20–1.40%. Finally, the developed quadruplex RT-qPCR was used to detect 1823 fecal samples collected in Guangxi Province, China between January 2024 and December 2024. The results indicated that the positivity rates of PSV, PKV, PTV, and EV-G were 15.25% (278/1823), 21.72% (396/1823), 18.82% (343/1823), and 27.10% (494/1823), respectively, and there existed phenomena of mixed infections. Compared with the reference RT-qPCR/RT-PCR established for these four viruses, the coincidence rates for the detection results of PSV, PKV, PTV, and EV-G reached 99.51%, 99.40%, 99.51%, and 99.01%, respectively. In conclusions, the developed quadruplex RT-qPCR could simultaneously detect PSV, PKV, PTV, and EV-G, and provided an efficient and convenient detection method to monitor the epidemic status and variation of these viruses. Full article

African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5′ untranslated region (5′UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV. Full article

Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022–2024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 × 10² copies/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19–1.31% and inter-assay variability of 0.10–0.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145/1688), 8.65% (146/1688), 2.84% (48/1688), and 1.30% (22/1688), respectively, while the positivity rates using the reference assays were 8.47% (143/1688), 8.53% (144/1688), 2.78% (47/1688), and 1.24% (21/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV. Full article

Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses. The results demonstrated that the assay effectively tested PoAstV, PoSaV, PoNoV, and PoRVA without cross-reactivity with other swine viruses, and had limits of detection (LODs) of 138.001, 135.167, 140.732, and 132.199 (copies/reaction) for PoAstV, PoSaV, PoNoV, and PoRVA, respectively, exhibiting high specificity and sensitivity. Additionally, it displayed good reproducibility, with coefficients of variation (CVs) of 0.09–1.24% for intra-assay and 0.08–1.03% for inter-assay. The 1578 clinical fecal samples from 14 cities in Guangxi Province, China, were analyzed via the developed assay. The results indicated that the clinical samples from Guangxi Province exhibited the prevalence of PoAstV (35.93%, 567/1578), PoSaV (8.37%, 132/1578), PoNoV (2.98%, 47/1578), and PoRVA (14.32%, 226/1578), and had a notable incidence of mixed infections of 18.31% (289/1578). Simultaneously, the 1578 clinical samples were analyzed with the previously established assays, and the coincidence rates of these two approaches exceeded 99.43%. This study developed an efficient and precise diagnostic method for the detection and differentiation of PoAstV, PoSaV, PoNoV, and PoRVA, enabling the successful diagnosis of these four diseases. Full article

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, and dehydration. Four pairs of primers and probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, and FRV NSP4 genes, and a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable of the simultaneous detection of four feline enteroviruses was developed after optimization of reaction conditions. The established quadruplex RT-qPCR assay showed high specificity, sensitivity, and reproducibility. The assay could detect and discriminate FeKoV, FeAstV, FeBuV, and FRV, but not other feline-related pathogens. The limits of detection (LODs) of FeKoV, FeAstV, FeBuV, and FRV were 109.761, 115.834, 125.481, and 113.875 copies/reaction, respectively. The intra- and inter-assay coefficients of variation (CV) were 0.15–1.61% and 0.15–1.59%, respectively. In all, 1869 clinical samples from Guangxi province in Southern China were tested using the developed assay, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93%, 9.36%, 0.32%, and 0.75%, respectively. These samples were also tested using reference assays, and the coincidence rates of the results between the developed and reference methods were 99.63% (FeKoV), 98.72% (FeAstV), 100% (FeBuV), and 100% (FRV), respectively. The results indicated that the developed assay could provide a new detection method for these four viruses associated with feline gastroenteritis. Full article

The ongoing COVID-19 pandemic, caused by the rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since early 2020, has highlighted the need for sensitive and reliable diagnostic methods. Droplet digital PCR (ddPCR) has demonstrated superior performance over the gold-standard reverse transcription PCR (RT-PCR) in detecting SARS-CoV-2. In this study, we explored the development of a multiplex ddPCR assay that enables sensitive quantification of SARS-CoV-2, which could be utilized for antiviral screening and the monitoring of COVID-19 patients. We designed a quadruplex ddPCR assay targeting four SARS-CoV-2 genes and evaluated its performance in terms of specificity, sensitivity, linearity, reproducibility, and precision using a two-color ddPCR detection system. The results showed that the quadruplex assay had comparable limits of detection and accuracy to the simplex ddPCR assays. Importantly, the quadruplex assay demonstrated significantly improved performance for samples with low viral loads and ambiguous results compared to the standard qRT-PCR approach. The developed multiplex ddPCR represents a valuable alternative and complementary tool for the diagnosis of SARS-CoV-2 and potentially other pathogens in various application scenarios beyond the current COVID-19 pandemic. The improved sensitivity and reliability of this assay could contribute to more effective disease monitoring and antiviral screening during the ongoing public health crisis. Full article

Background: Canine coronavirus (CCoV), canine rotavirus (CRV), canine parvovirus (CPV), and canine distemper virus (CDV) cause gastroenteritis in dogs, and co-infections of these pathogens are common in China. In particular, CCoV and CRV are confirmed to have important zoonotic potential and cause public health issues. It is difficult to diagnose these diseases based only on clinical manifestations and pathological damage. Methods: In this study, four pairs of specific primers and probes targeting the CCoV M, CRV VP7, CPV VP2, and CDV N genes were designed. The reaction conditions, including the primer and probe concentrations, annealing temperatures, and reaction cycles, were optimized for the development of a quadruplex RT-qPCR for the detection of CCoV, CRV, CPV, and CDV. The assay was used to test 1028 clinical samples to validate its application. Results: A quadruplex RT-qPCR was successfully established for the differential detection of CCoV, CRV, CPV, and CDV, with good specificity, high sensitivity, and excellent repeatability. The assay could specifically detect CCoV, CRV, CPV, and CDV without cross-reactivity with the other canine viruses tested. It showed high sensitivity with limits of detection (LOD) of 1.1 × 10² copies/reaction for all four plasmid constructs. It showed excellent repeatability, with 0.05–0.90% intra-assay variation and 0.02–0.94% inter-assay variation. The 1028 clinical samples were tested using the quadruplex RT-qPCR and a reported reference RT-qPCR. The positivity rates of CCoV, CRV, CPV, and CDV were 9.53%, 0.97%, 25.68%, and 5.06% using the developed assay, and 9.05%, 0.88%, 25.68%, and 4.86% using the reference assay, with agreements higher than 99.32%. Conclusion: The results indicated that a rapid and accurate quadruplex RT-qPCR was developed for the detection and differentiation of CCoV, CRV, CPV, and CDV. Full article

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis. Full article

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15–1.14% and inter-assay CVs of 0.07–0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China. Full article

Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan^® RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (p-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission. Full article

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/μL (final reaction concentration of 12.1 copies/μL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses. Full article

Guanine-rich DNA sequences tending to adopt noncanonical G-quadruplex (G4) structures are over-represented in promoter regions of oncogenes. Ligands recognizing G4 were shown to stabilize these DNA structures and drive their formation regulating expression of corresponding genes. We studied the interaction of several plant secondary metabolites (PSMs) with G4s and their effects on gene expression in a cellular context. The binding of PSMs with G4s formed by the sequences of well-studied oncogene promoters and telomeric repeats was evaluated using a fluorescent indicator displacement assay. c-MYC G4 folding topology and thermal stability, as well as the PMS influence on these parameters, were demonstrated by UV-spectroscopy and circular dichroism. The effects of promising PSMs on c-MYC expression were assessed using luciferase reporter assay and qPR-PCR in cancer and immortalized cultured cells. The ability of PMS to multi-targeting cell signaling pathways was analyzed by the pathway-focused gene expression profiling with qRT-PCR. The multi-target activity of a number of PSMs was demonstrated by their interaction with a set of G4s mimicking those formed in the human genome. We have shown a direct G4-mediated down regulation of c-MYC expression by sanguinarine, quercetin, kaempferol, and thymoquinone; these effects being modulated by PSM’s indirect influence via cell signaling pathways. Full article

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4_mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4_mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4_mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt², qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics. Full article