Search Results (1,049)

Cacao leafroll virus (CaLRV; species Polerovirus CALRV, family Solemoviridae), previously referred to as cacao polerovirus, infects cacao (Theobroma cacao) germplasm in the Americas. Screening of 287 cacao accessions with three published multiplex RT-PCR assays detected CaLRV in nine accessions but produced reproducibly discordant results for the accession LCT EEN 412. Metatranscriptomic sequencing generated three near-complete CaLRV genome sequences, CaLRV-TSAN792, CaLRV-POUND15A, and CaLRV-LCTEEN412 (5985–5989 nt), from accessions originating from Brazil, Trinidad and Tobago, and Ecuador, respectively. The divergent CaLRV-LCTEEN412 assembly was verified by RT-PCR, cloning, and nanopore sequencing. In addition, analysis of five public cacao RNA-seq datasets yielded one near-complete CaLRV-PA16 assembly included in whole-genome analysis and four fragmented assemblies that informed primer design. Comparison of the four sequences characterised here with six previously reported genomes showed that nine shared 98.8–99.7% pairwise nucleotide identity, whereas CaLRV-LCTEEN412 shared 85.0–85.2%. Primer-binding-site mismatches in CaLRV-LCTEEN412 were consistent with its discordant detection by published assays. Two new multiplex RT-PCR assays were therefore designed from the expanded alignment and evaluated using four CaLRV-positive cacao accessions, including LCT EEN 412. These results expand known CaLRV genomic diversity and provide dual-target tools that reduce dependence on a single locus and enable recognition of target discordance. Full article

This study aimed to explore whether zona-associated residual sperm may contribute to exogenous Y-DNAY-DNA contamination and cause sex bias in PCR-based sex classification of ovine early-stage embryos, based on the amplification of the AMEL gene. Primers targeting the conserved regions of the ovine AMELX and AMELY genes were designed, and a nested PCR system using AMEL and YWHAZ genes was established. Group A comprised 17 female and 33 male embryos, corresponding to a female-to-male ratio of 0.52:1, whereas Group B comprised 23 female and 27 male embryos, corresponding to a female-to-male ratio of 0.85:1. DAPI staining revealed sperm-morphology structures or structures containing sperm-derived DNA adjacent to the zona pellucida of ovine early-stage embryos. Sequence alignment of PCR amplicons showed consistency with the AMELX and AMELY reference genes. In conclusion, these results support our research hypothesis that zona-associated residual sperm may serve as a potential source of Y-DNAY-DNA contamination and interfere with PCR-based embryonic sex determination. Full article

Carbapenem resistance in Pseudomonas aeruginosa is frequently mediated by the mutational inactivation or complete loss of the oprD porin gene. However, extreme genetic polymorphism within oprD limits traditional PCR amplification, a bottleneck for molecular surveillance. This study aimed to design and validate a novel quantitative PCR (qPCR) primer set flanking the 5′ and 3′ untranslated regions to reliably amplify the complete oprD cassettes across divergent clinical isolates. The assay demonstrated optimal amplification at 54 °C and an analytical limit of detection (LoD) between 10⁻⁴ and 10⁻⁵ ng of genomic DNA mass. This assay successfully matched 94.09% (1608/1709) of globally distributed P. aeruginosa strains via in silico BLASTn analysis. In a blinded validation cohort of 100 clinical isolates (50 P. aeruginosa and 50 non-P. aeruginosa controls), the qPCR assay achieved 100.0% analytical specificity without cross-reactivity. Among the 50 P. aeruginosa isolates including 40 carbapenem-resistant (CRPA) and 10 carbapenem-susceptible (CSPA), the qPCR assay exhibited 96.0% sensitivity, successfully amplifying oprD cassettes in 95.0% (38/40) of CRPA and 100.0% (10/10) of CSPA strains without phenotypic bias (p > 0.9999). Notably, when combined with sequencing, the two qPCR-negative CRPA isolates were confirmed to harbor true biological oprD deletions. This optimized assay provides a robust molecular surveillance tool for detecting oprD cassettes to facilitate the epidemiological genetic characterization in both CRPA and CSPA isolates. Full article

Quantitative real-time PCR (qRT-PCR) is a widely used technique for quantifying gene expression. However, accurate data normalization requires stable reference genes that exhibit constant expression levels across different experimental conditions. To date, no suitable reference genes have been validated for atemoya fruits (Annona cherimola Mill × A. squamosa L.). This study aimed to evaluate the expression stability of candidate reference genes in atemoya across distinct tissues, postharvest storage stages, and various treatment conditions. To this end, we designed specific primers for six reference genes: ubiquitin carrier-like protein (UBC), actin 7 (ACT7), actin 11 (ACT11), elongation factor 1α (EF1α), 18S ribosomal RNA (18S), and β-tubulin (TUB). Total RNA was extracted from atemoya tissues (e.g., petals, young fruits, and roots), for pulp across three post-harvest time points (0, 2, and 4 days) and under different conditions: 28 °C, 15 °C, ethylene, or 1-MCP treatment. The geNorm tool was subsequently used to identify the most stable reference transcripts under each experimental condition. ACT7 was the most stable reference gene across atemoya tissues, while UBC ranked first under all postharvest conditions tested (28 °C storage, 15 °C cold storage, ethylene, and 1-MCP). Furthermore, the suitability of these reference genes under ethylene and 1-MCP treatments was validated by examining the expression patterns of AaPG and AaERF. Collectively, the combined use of ACT7, UBC, and 18S proved to be the most reliable normalization strategy across all experimental conditions. This validated multi-gene approach provides a robust foundation for future investigations into gene expression and the postharvest ripening mechanisms of atemoya. Full article

Mulberry mosaic dwarf-associated virus (MMDaV), a member of the genus Mulcrilevirus in the family Geminiviridae, poses a serious threat to mulberry cultivation and growth in China. Early and accurate diagnosis is a prerequisite for the effective prevention and control of MMDaV-related disease. In this study, a loop-mediated isothermal amplification (LAMP)-based detection method was established for the specific identification of MMDaV. Three pairs of specific primers were designed targeting the nucleotide sequences of the MMDaV V2 gene. The optimized LAMP reaction was performed at a constant temperature of 65 °C for 60 min, and the established method exhibited high specificity with no cross-reactivity observed against other tested geminiviruses. The LAMP method achieved a detection limit of 200 pg of target MMDaV DNA, which was tenfold more sensitive than conventional PCR. Furthermore, the amplification results could be directly visualized via distinct color change. Collectively, the developed LAMP method enables efficient, sensitive, and specific isothermal detection of MMDaV and holds great promise for rapid diagnosis of mulberry viral disease. Full article

Background/Objectives: Direct bonding of orthodontic brackets is standard in fixed-appliance therapy, but clinical success depends on achieving reliable bond strength without compromising enamel integrity during debonding. This in vitro study evaluated whether applying a metal primer to stainless-steel bracket bases affects shear bond strength (SBS) to enamel, and whether primer coverage and bracket-base design influence adhesive-remnant distribution and enamel integrity. Methods: A total of 132 extracted human maxillary premolars were randomly allocated to six groups (n = 22 each) defined by bracket base design (mesh or integral) and primer coverage (none, full, or partial central coverage). Brackets were bonded with Transbond XT, and Reliance Metal Primer was applied according to group allocation. After 2000 thermal cycles (5–55 °C), specimens were debonded in shear at 1 mm/min. SBS was calculated, adhesive remnants were quantified with ImageJ and scored using the adhesive remnant index (ARI), and enamel damage was assessed using an enamel damage index (EDI). Results: Mean SBS values were 15.68 MPa (MC), 18.38 MPa (MF), 20.89 MPa (MP), 13.04 MPa (IC), 17.11 MPa (IF), and 12.66 MPa (IP). Two-way ANOVA showed significant main effects of bracket base design (F(1, 126) = 16.06, p < 0.001, partial η² = 0.113) and primer coverage (F(2, 126) = 3.98, p = 0.021, partial η² = 0.059), together with a significant bracket-base-design × primer-coverage interaction (F(2, 126) = 4.46, p = 0.014, partial η² = 0.066). ARI distributions differed significantly across the six experimental groups (χ²(15) = 75.24, p < 0.001, Cramér’s V = 0.436), whereas EDI scores did not differ significantly among groups (p = 0.756). Conclusions: The effect of the metal primer on SBS depended on bracket base design and primer coverage. Partial coverage produced the highest SBS among mesh-base brackets and was significantly higher than the mesh control. Among integral-base brackets, full coverage yielded the highest numerical SBS, but within-design pairwise differences were not significant. ARI failure patterns also varied significantly across the six experimental groups, whereas no significant difference in stereomicroscopically detectable enamel damage was observed. Full article

Mannheimia haemolytica is a primary pathogen causing bacterial pneumonia in cattle and sheep. Multiple PCR targets exist for its identification, but no standardized primer pairs have been widely adopted. This study evaluated the universality of six reported primers (including rpt2) using clinical M. haemolytica isolates. Genus specificity was tested against standard strains of common ruminant pathogenic/commensal bacteria and other Mannheimia species. Four primer sets (gcp, artJ-lktC, lktD, sodA) failed to distinguish M. haemolytica from other Mannheimia species. The rpt2 primer showed suboptimal universality, with 3 of 22 tested isolates yielding negative results. The rpoB primer had favorable universality and specificity, but its 136 bp amplicon was unsuitable for constructing multiplex PCR with M. haemolytica serotyping primers. We identified design flaws in the original rpt2 primer and developed a novel rpt2-N primer. Evaluation confirmed its strong universality, specificity, and sensitivity, as well as compatibility with serotyping primers for multiplex PCR assays. Field testing on three goat farms in the Chongqing region showed markedly higher M. haemolytica positivity rates after sharp temperature drops and transportation. Serotype A2 was confirmed as the dominant serotype in goats in Chongqing. This study systematically assessed multiple PCR identification primers, confirmed the feasibility of rpoB, and resolved the universality limitation of rpt2, providing technical support for rapid and accurate M. haemolytica detection. Full article

The Zika virus (ZIKV) remains an important public health concern owing to its association with neurological disorders and congenital abnormalities. Genetically, ZIKV is classified into two major lineages—African and Asian—and continues to evolve, with evidence of genetic diversification following its introduction into the Americas. The study of the envelope (E) gene of ZIKV is a key tool for understanding viral biology, phylogenetic analysis, and developing antiviral drugs and vaccine candidates. However, few studies have focused on designing primers capable of efficiently detecting and amplifying this essential gene. In this study, we developed universal primer sets capable of amplifying and sequencing the full-length ZIKV E gene across genetically diverse lineages and strains. Primers were designed based on representative ZIKV sequences available in public databases and validated using viral isolates and in vitro-spiked plasma samples. The assay demonstrated high specificity and analytical sensitivity, with the analytical detection limit established using serially diluted DNA amplicons from the target region. Furthermore, ZIKV was successfully detected in plasma samples spiked at 1 × 10³ PFU/mL, which enabled complete sequencing of the E gene. These findings highlight the utility of our primers for molecular epidemiological studies, strengthening arbovirus surveillance and improving diagnostic capacity in regions where multiple arboviruses co-circulate, using a simple and reliable assay. Full article

Short DNA barcodes may facilitate sequence recovery from degraded material, but their ability to retain target-family identity while excluding related taxa varies among genomic regions. We computationally evaluated 16 nuclear, plastid, and mitochondrial marker regions from 11 Asterales families using 279,956 NCBI locus–record matches and an accession-disjoint discovery/test design. Thirty-one candidate segments of 50–200 bp (mean, 98.55 bp) were screened in discovery data and evaluated for within-Asteraceae sequence recall, differentiation from non-Asteraceae Asterales, in silico primer behavior, phylogenetic placement, and exploratory matching across 808 metadata-defined metagenomic samples. Conserved regions such as matR and rbcL showed high within-Asteraceae identity, whereas ITS1, ITS, and trnH-psbA showed larger differences from related-family backgrounds; ITS2 and ycf1 showed intermediate profiles. Candidate segments were placed within or immediately adjacent to Asteraceae reference branches in segment-specific maximum-likelihood analyses, although support and topology varied among regions. Metadata-defined target-containing groups had higher mean query coverage and identity than background groups; because target presence was not independently verified and no classifier was fitted, these comparisons were descriptive and did not estimate diagnostic accuracy. Definitionally linked sequence statistics were interpreted as structural associations rather than evidence of causal evolutionary mechanisms. These results provide a family-level computational comparison of candidate short segments for Asteraceae detection within Asterales. Species identification, operational marker combinations, threshold robustness, and laboratory performance require validation using taxonomically dense, voucher-linked, and experimentally characterized datasets. Full article

Grapevine leafroll-associated virus 3 (GLRaV3) and grapevine red blotch virus (GRBV) are two major pathogens associated with significant economic losses, requiring reliable and sensitive diagnostic tools for rapid identification. Current RT-qPCR and qPCR assays are widely used in certification programs; however, the risk of false-negative results due to the genetic diversity of plant viruses is always a concern. Here, we present a new targeted HiPlex assay designed for the detection of GLRaV3 and GRBV. A custom primer panel containing 98 oligonucleotides targeting GLRaV3 and 14 targeting GRBV was evaluated across 45 different grapevine accessions. HiPlex diagnostic performance metrics were evaluated up to the 1:1000 dilution level. The HiPlex assay detected both viruses in 30 ng of total nucleic acids (TNA) and at 1:10 and 1:100 dilutions, exhibiting a strong inverse correlation between read counts and Ct values. Analysis of reads from qPCR-negative samples allowed the determination of a positivity cutoff of 5000 reads, which provided high specificity while maintaining sensitivity. The results presented here support the application of HiPlex amplicon sequencing for the detection of GLRaV3 and GRBV and provides a foundation for future expansion of the primer panel to additional grapevine viruses. To our knowledge, this is the first study evaluating multiplex PCR-based amplicon sequencing specifically for grapevine virus diagnostics. Full article

Cynoglossus semilaevis papillomavirus (CsPaV) is an emerging viral pathogen associated with high mortality in farmed Chinese tongue soles. A sensitive, specific, and quantitative method is needed for CsPaV surveillance and epidemiological studies. In this study, we designed primers and a probe that targeted the CsPaV L1 gene. We then established a TaqMan probe-based quantitative PCR (qPCR) assay. The assay detected as few as 5.8 × 10¹ copies/µL and was 1000-fold more sensitive than conventional PCR. The assay did not cross-react with the other aquatic pathogens tested. The intra-assay coefficients of variation ranged from 0.23% to 0.84%, and the inter-assay coefficients ranged from 0.56% to 1.48%. These results showed that the assay had good repeatability. We used the assay to study the tissue distribution and temporal dynamics of CsPaV after experimental infection. Healthy Chinese tongue soles received an intraperitoneal injection of 300 µL of inoculum containing 3.66 × 10⁵ CsPaV copies/µL. Three fish were sampled at 1, 2, 3, 5, and 7 days post-infection. CsPaV DNA was detected in all tissues examined. The kidney had the highest viral DNA load, which reached 2.47 × 10⁷ copies/mg at 7 days post-infection. Among 176 clinically diseased Chinese tongue soles collected between 2023 and 2026, 80.68% tested positive for CsPaV. CsPaV DNA was also detected in several other aquatic species. These findings provide a sensitive tool for CsPaV surveillance and molecular epidemiological studies. The assay may also support disease prevention and control in Chinese tongue sole aquaculture. Full article

The development of effective conservation and management strategies for wild Ribes populations requires a comprehensive understanding of genetic diversity and population structure of these taxa, but only a few genetic studies exist, and these have focused on cultivated material in the genus. We evaluated the use of MobiSeq as a reduced representation sequencing approach to detect genetic variation in three Ribes species, and we used the data to characterize the genetic diversity and population structure in the Netherlands and the relationships between the wild Ribes populations, the ex situ genebank collection, and cultivars. MobiSeq with a single primer designed for repetitive elements that occur in the three species produced 17,000–31,000 SNPs per species after filtering against markers with minor allele frequency (MAF) < 0.01 and markers deviating from Hardy-Weinberg equilibrium (HWE). The SNPs were called against consensus sequences, producing separate sets of markers for each of the species. All three species had moderate genetic diversity (H_S = 0.141–0.239) and relatively low differentiation (F_ST = 0.049–0.083) with no evidence of inbreeding, consistent with their predominantly outcrossing life-history traits. This suggests that Dutch wild populations have largely retained their genetic diversity despite the current habitat fragmentation. Several putatively wild individuals showed evidence of cultivated ancestry, possibly indicating historical introgression or naturalized escapes from cultivation, particularly in R. rubrum and R. uva-crispa. Low differentiation between in situ and ex situ material (F_ST = 0.004–0.026) suggests that the genebank collections already capture most of the genetic diversity present in wild populations. Full article

To achieve rapid, accurate, and quantitative detection of peste des petits ruminants virus (PPRV), we aligned the available PPRV genomic sequences, screened for conserved regions, and designed specific primers and probes accordingly. An RT-droplet digital PCR (RT-ddPCR) method was established, and a series of reaction conditions were optimized. Subsequently, the specificity, sensitivity, and repeatability of the method were subsequently evaluated. The results demonstrated that the RT-ddPCR method exhibited good specificity, with positive results obtained exclusively for PPRV and negative results for all other goat-derived viral or bacterial biological products and seed viruses tested. The method showed promising analytical sensitivity, with a LOD of 1.0 copies/μL for gene copy number. The method also demonstrated good repeatability, with coefficients of variation in all groups not exceeding 5%. In a preliminary clinical evaluation using 150 field samples, RT-ddPCR identified 113 positive cases, compared to 105 by RT-qPCR, with 8 additional low-copy-number positives detected exclusively by RT-ddPCR, and although the sample size is limited for precise diagnostic performance estimation, the assay showed 100% relative diagnostic sensitivity (105/105) and 82.2% relative diagnostic specificity (37/45), with almost perfect agreement between the two methods (κ = 0.87). As the positive signals in these discordant samples were too weak for independent confirmation by Sanger sequencing, they should be interpreted as “presumptive positives” requiring further investigation. The RT-ddPCR method established in this study enables sensitive and rapid detection of PPRV nucleic acid. However, detection of low-copy-number targets should be interpreted with caution, as it does not necessarily indicate active infection or epidemiologically significant virus circulation. This assay shows promise as a valuable research and surveillance tool for PPRV nucleic acid detection, particularly for confirming low-level viral RNA in samples near the detection limit of conventional methods, and could complement existing RT-qPCR-based approaches in PPR eradication programs. Full article

To establish a rapid nucleic acid detection method for field screening of Brucella spp., a visual assay based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a was developed. Four pairs of RPA primers and two crRNAs were designed according to the conserved Brucella bcsp31 sequence. The optimal RPA primer–crRNA combination was screened using agarose gel electrophoresis and fluorescence readout, and the concentrations of Cas12a protein, crRNA, and ssDNA fluorescent reporter were optimized. RPA primer pair 2 showed the best amplification performance, and RPA2-crRNA1 was selected as the optimal combination for the assay. The final reaction conditions were 200 nM Cas12a protein, 200 nM crRNA, and 250 nM ssDNA fluorescent reporter. The limit of detection of the established method was 2.47 × 10⁰ copies/μL. No cross-reactivity was observed with Mycoplasma capricolum subsp. capripneumoniae, Chlamydia abortus, Chlamydia psittaci, or Salmonella enterica serovar Typhimurium. Among 56 clinical sheep nasal swab samples collected during routine surveillance, qPCR identified 22 positive and 34 negative samples, while the RPA-CRISPR/Cas12a assay identified 20 positive and 36 negative samples. The positive agreement, negative agreement, overall agreement, positive predictive value, negative predictive value, and Cohen’s kappa value were 90.91%, 100.00%, 96.43%, 100.00%, 94.44%, and 0.924, respectively. These findings provide preliminary evidence that the developed bcsp31-targeted RPA-CRISPR/Cas12a assay is rapid and visually interpretable and may be useful for preliminary Brucella spp. screening in routine veterinary surveillance settings. Larger-scale validation using different sample types and samples from different regions is still required. Full article

Lumpy skin disease (LSD) is a transboundary viral disease of cattle, including Asian water buffalo and yaks, and certain wild ruminants (e.g., African buffalo, giraffe, wildebeest, eland, and Arabian oryx). It is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus (family Poxviridae) together with goatpox virus (GTPV) and sheeppox virus (SPPV). High nucleotide identity and serological cross-reactivity among these viruses hinder differential diagnosis. The aim of this study was to develop a Duplex TaqMan-MGB qPCR Assay for Differential Detection of Chinese Epidemic Lumpy Skin Disease Virus Strains and Goatpox Virus. We developed a duplex TaqMan-MGB real-time PCR (qPCR) assay targeting the LSDV GPCR and GTPV RPO30 loci. Virus-specific primers and MGB probes were designed, and the reaction was optimized for single-tube, two-target detection. The assay showed no cross-amplification, limits of detection of 1 × 10¹ copies/μL (LSDV) and 1 × 10¹ copies/μL (GTPV), and coefficients of variation < 1%. The assay was applied to 175 yak-derived field specimens from Qinghai–Tibet Plateau, of which 16 and 36 were positive for LSDV and GTPV, respectively. LSDV- and GTPV-positive samples showed specific amplification in the FAM and VIC channels, respectively. The duplex format enables concurrent detection and unambiguous differentiation of LSDV and GTPV and is compatible with high-throughput screening. This sensitive, specific, and reproducible assay supports surveillance and control of LSD in endemic and at-risk regions. Full article