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Results for “multiplex real-time PCR”

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PubMed2026

Enhancing Hemoglobin Bart's hydrops fetalis syndrome prevention: a single-tube multiplex real-time PCR assay for the comprehensive detection of four significant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA) found in Thailand.

BACKGROUND: Hemoglobin (Hb) Bart's hydrops fetalis is a major public health concern in Southeast Asia, particularly in Thailand. Current screening strategies target the two most common α0 -thalassemia deletions (--SEA and --THAI). METHOD: In this study, we developed a single-tube multiplex real-time PCR assay for the simultaneous detection of four clinically relevant α0-thalassemia deletions (--SEA, --THAI, --CR, and --SA). The assay was validated using 538 clinical samples with diverse thalassemia genotypes and compared against conventional gap-PCR as the reference method. Analytical performance, including sensitivity, specificity, and limit of detection (LOD), was evaluated. In addition, clinical utility was assessed in 22 prenatal diagnosis cases at risk of Hb Bart's hydrops fetalis. RESULTS: The study cohort demonstrated substantial genetic heterogeneity, comprising 43 distinct genotypes. The developed assay achieved 100% sensitivity and specificity for all targeted deletions, with complete concordance with gap-PCR results. No cross-reactivity was observed with α+-thalassemia. The assay demonstrated a high analytical sensitivity with a LOD of 9.76 × 10-3 ng per reaction. Whereas in prenatal diagnosis, all 22 fetal genotypes were accurately identified, including five cases of homozygous --SEA and one rare compound heterozygous --SEA/--CR fetus. CONCLUSIONS: This study presents a rapid, accurate, and cost-effective multiplex real-time PCR assay capable of detecting both common and rare α0-thalassemia deletions in a single reaction. The assay demonstrates strong potential for implementation in routine clinical laboratories and large-scale population screening, contributing to improved prevention and control of severe thalassemia syndromes in high-prevalence regions.

Humans↗

PubMed2025

Rapid and accurate sepsis diagnostics via a novel probe-based multiplex real-time PCR system.

Sepsis is a critical clinical emergency that requires prompt diagnosis and intervention. Its prevalence has increased due to the aging population and increased antibiotic resistance. Early identification and the use of innovative technologies are crucial for improving patient outcomes. Modern methodologies are needed to minimize the turnaround time for diagnosis and improve outcomes. Rapid diagnostic tests and multiplex PCR are effective but have limitations in identifying a range of pathogens and target genes. Our study evaluated two novel probe-based multiplex real-time PCR systems: the SEPSI ID and SEPSI DR panels. These systems can quickly identify bacterial and fungal pathogens, alongside antibiotic resistance genes. The assays cover 29 microorganisms (gram-negative bacteria, gram-positive bacteria, yeast, and mold species), alongside 23 resistance genes and four virulence factors. A streamlined workflow uses 2 µL of broth from positive blood cultures (BCs) without nucleic acid extraction and provides results in approximately 1 h. We present the results from an evaluation of 228 BCs and 22 isolates previously characterized by whole-genome sequencing. In comparison to the reference methods, the SEPSI ID panel demonstrated a sensitivity of 96.88%, a specificity of 100%, and a PPV of 100%, whereas the SEPSI DR panel showed a sensitivity of 97.8%, a PPV of 89.7%, and a specificity of 96.7%. Both panels also identified additional pathogens and resistance-related targets not detected by conventional methods. This assay shows promise for rapidly and accurately diagnosing sepsis. Future studies should validate its performance in various clinical settings to enhance sepsis management and improve patient outcomes.IMPORTANCEWe present a new diagnostic method that enables the quick and precise identification of pathogens and resistance genes from positive blood cultures, eliminating the need for nucleic acid extraction. This technique can also be used on fresh pathogen cultures. It has the potential to greatly improve treatment protocols, leading to better patient outcomes, more responsible antibiotic use, and more efficient management of healthcare resources.

Humans↗

PubMed2025

KpSC-ID: a multiplex real-time PCR assay for the simultaneous detection of the Klebsiella pneumoniae species complex and specific identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae and Klebsiella variicola.

The Klebsiella pneumoniae species complex (KpSC) comprises five closely related bacterial species, namely Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Klebsiella quasivariicola and Klebsiella africana. The KpSC is ubiquitous in the environment and is also an important human pathogen, particularly associated with healthcare-associated infections. The accurate detection and differentiation of the KpSC is challenging owing to the close phenotypic and genotypic identity (93-95% average nucleotide identity) shared between these members. Current diagnostic assays either fail to detect and identify all KpSC members or misidentify some KpSC members as K. pneumoniae sensu stricto. It is currently estimated that ~20% of human infections are caused by members of the KpSC other than K. pneumoniae. This leads to underreporting of some KpSC members in both clinical and environmental settings, which impacts our understanding of the importance of each species. Furthermore, it limits our understanding of the global and local epidemiological impact of some members of the KpSC. In this study, a rapid multiplex real-time PCR assay (KpSC-ID) was designed and developed to detect all KpSC members while simultaneously identifying the predominant human pathogens K. pneumoniae, K. quasipneumoniae and K. variicola. Assay performance was verified in silico using a panel of over 1,000 publicly available genome sequences and experimentally validated using a panel of genomic DNA extracted from 54 Enterobacteriaceae. The assay displayed excellent specificity against over 1,000 genome sequences tested in silico. During in vitro validation, the pan-KpSC assay detected each (29/29) KpSC species and strains tested. For the species-specific assays, 100% specificity was demonstrated in the K. pneumoniae, K. quasipneumoniae and K. variicola assays, respectively. Sensitivity of 10 genomic equivalents was demonstrated for each assay. Ultimately, the diagnostic assay developed in this study can improve our understanding of the significance of KpSC members, which is important when investigating their routes of transmission and epidemiology.

Klebsiella↗

PubMed2019

Development of a multiplex real-time RT-PCR assay for simultaneous detection and differentiation of influenza A, B, C, and D viruses.

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.

Animals↗

PubMed2026

Development and epidemiological investigation of a TaqMan-based multiplex real-time quantitative PCR assay for simultaneous detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV).

INRODUCTION: Infectious diseases caused by bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine norovirus (BNoV), bovine enterovirus (BEV), and bovine coronavirus (BCoV) significantly threaten the cattle industry, resulting in substantial economic losses. These pathogens often present similar clinical signs, such as diarrhea, vomiting, and reproductive disorders in pregnant cattle, and frequent covert or mixed infections further complicate accurate diagnosis. Therefore, rapid, sensitive, and field‑deployable diagnostic methods are essential for effective disease surveillance and control in the cattle industry. METHODS: In this study, we report for the first time the establishment of a TaqMan‑based real‑time quantitative PCR (qPCR) assay that enables simultaneous detection of these five bovine viruses. Multiple sequence alignment of conserved genomic regions was performed, and virus‑specific primers and probes were designed and optimized using Beacon Designer 7 software. Subsequently, a TaqMan‑based multiplex real‑time qPCR assay was established for simultaneous detection of BVDV, AKAV, BNoV, BEV, and BCoV. The established detection method was applied to 200 clinical samples collected from 10 farms in multiple regions of Jilin Province. RESULTS: The results showed that the detection rates for BVDV, AKAV, BNoV, BEV, and BCoV were 33.50%, 0.50%, 4.50%, 7.50%, and 12.00%, respectively. Mixed infections were detected in 9 samples co‑infected with two of the five pathogens, with an overall mixed infection rate of 4.50%. Compared with conventional PCR, coincidence rates were 100% for BVDV, AKAV, BNoV, BEV, and BCoV. DISCUSSION: These findings indicate that the TaqMan multiplex real‑time qPCR assay developed here demonstrates favorable specificity, sensitivity, and reproducibility. This assay enables efficient detection and surveillance of bovine viruses, offering a reliable technical tool for the diagnosis and control of corresponding viral diseases in cattle.

Akabane virus (AKAV)↗

PubMed2026

Human papillomavirus viral load as promising surrogate biomarker of cervical cancer risk and clinical outcome.

INTRODUCTION: Persistent high-risk human papillomavirus (HR-HPV) causes cervical precancerous lesions and cancer. While molecular HPV DNA testing offers superior sensitivity over cytology as a primary screening method, its limited specificity leads to unnecessary follow-up procedures. Therefore, identifying surrogate biomarkers to distinguish transient infections from clinically relevant, persistent ones is essential for improving risk stratification. A comprehensive literature search across PubMed/MEDLINE, Embase, Scopus, and Web of Science databases up to December 2025 identified studies evaluating HR-HPV viral load in cervical lesion progression. AREAS COVERED: Oncogenic HPV viral load, the quantity of HPV genomes in a sample, is a promising biomarker. Levels correlated positively with HR-HPV persistence, increasing the risk of high-grade lesions and invasive cervical cancer. Furthermore, quantification provides prognostic information regarding disease severity, therapeutic response, and post-treatment recurrence. EXPERT OPINION: Recent standardization and validation of multiplex real-time PCR techniques supports integrating HPV viral load into clinical pratice. Incorporating viral load assessment into screening and management algorithms could significantly enhance diagnostic precision, enable personalized follow-up, and guide therapeutic decisions for women with HR-HPV-associated cervical disease. Refining these protocols will ultimately minimize over-treatment while ensuring rigorous monitoring for high-risk patients.

Humans↗

PubMed2026

A rapid molecular assay for the detection of hypervirulent Klebsiella pneumoniae in the context of antimicrobial resistance surveillance.

Hypervirulent Klebsiella pneumoniae (hvKP) represents an emerging clinical and public-health concern, particularly as hypervirulence increasingly converges with multidrug resistance. Current diagnostic approaches rely on phenotypic assays, such as the string test, or on whole-genome sequencing (WGS), both of which have limitations in specificity, turnaround time, standardization, and feasibility for routine surveillance. To address this gap, we developed a multiplex real-time PCR assay targeting key hvKP-associated virulence loci, including siderophore systems, hypermucoviscosity regulators, and additional markers linked to invasive potential. The assay was evaluated on 110 K. pneumoniae clinical isolates and 9 positive blood cultures, using WGS and the string test as comparators. The molecular panel demonstrated high concordance with WGS for principal virulence determinants, correctly identifying all high-virulence (score 4) profiles, and most intermediate profiles. Against WGS, the assay yielded a sensitivity of 82% and a specificity of 73%; performance against the string test was 96% and 87%, respectively. Direct testing from blood culture pellets yielded results consistent with both WGS and DNA-based PCR for the limited number of targets detected, supporting the technical feasibility of this approach. However, broader validation is needed to confirm performance in this specimen type. Overall, this multiplex PCR assay provides a targeted molecular screening approach for the rapid identification of hvKP-associated virulence profiles. Its agreement with genomic data supports its potential utility as an accessible complement to WGS for hvKP surveillance, although further workflow optimization will be required before broader routine implementation.IMPORTANCEThe global emergence of hypervirulent and multidrug-resistant K. pneumoniae represents a major public-health threat, as the convergence of virulence and antimicrobial resistance dramatically limits therapeutic options and increases the likelihood of severe, invasive, and potentially untreatable infections. Rapid identification of essential virulence determinants is therefore critical for timely clinical management and for preventing onward transmission. However, current diagnostic approaches are either insufficiently sensitive or require substantial resources, limiting their routine use. By providing a rapid and targeted molecular assay capable of detecting the principal loci associated with hypervirulent K. pneumoniae and by demonstrating the preliminary feasibility of its use directly on blood culture pellets previously identified as Klebsiella spp. by MALDI-TOF MS, this work provides a pragmatic approach for early virulence profiling. Implementation of such assays can significantly enhance epidemiological surveillance, support tailored patient management, and reduce the spread of high-risk K. pneumoniae lineages in both community and healthcare environments.

Klebsiella pneumoniae↗

PubMed2026

Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.

Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.

CRISPR/Cas diagnostics↗

PubMed2026

Clinical and genomic characterization of Influenza A co-infection with SARS-CoV-2 and Influenza B: a respiratory surveillance study in Assam, India.

Influenza and SARS-CoV-2 are the primary contributors to seasonal respiratory infections and frequently co-circulate, creating significant health challenges. The present respiratory surveillance study was conducted in Dibrugarh, Assam, India from January 2025 to August 2025 to investigate the genomic characteristics of circulating viruses and identify potential co-infections. Overall, 4,948 respiratory samples were screened using multiplex real-time PCR, followed by subtyping of Influenza A and Influenza B. Next-generation sequencing (NGS) was performed in selected positives of SARS-CoV-2 and Influenza A. Genomic analysis included mutational profiling, phylogenetic analysis and N-glycosylation site prediction using bioinformatics tools. Two co-infection cases were detected: one involving Influenza A (H3N2) with SARS-CoV-2 (Omicron XFG lineage) and another involving Influenza A (H3N2) with Influenza B (Victoria lineage). Both patients experienced mild illness without hospitalisation. NGS revealed that the Influenza A (H3N2) viruses belonged to clade 3C.2a1b.2a.2a.3a.1 while SARS-CoV-2 sequence was classified under the Omicron XFG lineage. Mutational analysis of the HA gene showed several amino acid differences compared to the reference vaccine strain A/Darwin/6/2021. N-glycosylation analysis predicted conserved sites at positions 79, 181, 262, and 301 in all strains along with an additional predicted site at position 110 in both co-infection cases. Although the co-infection cases presented with mild clinical manifestations, the observed genomic variations indicate a potential role of co-infecting viruses in shaping viral evolution. Given the limited genomic data available from Northeast India, the study underscores the need for sustained large scale follow up and genomic surveillance to monitor emerging mutations and target future vaccine strategies.

Humans↗

PubMed2025

Human papillomavirus (HPV) genotypes extended prevalence in the female population from a city in Northern Chile.

BACKGROUND: Cervical cancer is primarily associated with the presence of human papillomavirus (HPV), with high-risk genotypes HPV-16 and HPV-18 being the focus of vaccination programs in developing countries such as Chile. Preventive screening for cervical cancer in women aged 25 to 64 years remains centered on cytological techniques and is primarily performed based on clinical suspicion of cervical lesions. However, extended screening for HPV genotypes using DNA amplification methods is not routinely applied to the Chilean female population yet. This study aimed to determine the prevalence of high- and low-risk HPV genotypes in women without known risk factors in a city in northern Chile. METHODS: Cervicovaginal brushing samples were obtained from 390 women from Antofagasta city, Northern Chile, aged between 25 and 64 years; genomic DNA was extracted, and multiplex real-time PCR analysis was used to identify a larger group of high- and low-risk HPV genotypes. RESULTS: Among 390 samples, HPV prevalence was 36.9%, of which 54.9% were high-risk genotypes, 18.7% were low-risk genotypes, and 26.4% showed mixed infection with both high- and low-risk genotypes. High-risk genotypes 16, 58, 39, and 31 were the most frequently identified among HPV-positive samples. Furthermore, a significant association was observed between HPV presence and both age and suspicion of cervical alteration, and women testing positive for other sexually transmitted infections (STIs) were more likely to acquire HPV. CONCLUSIONS: Implementing a screening program that incorporates extended HPV genotyping in Chile, including testing for high-risk genotypes 16, 18, 31, 39, and 58, is crucial to optimize control, early detection, and vaccination efforts for Chilean circulating HPV genotypes that are not covered by the actual vaccine, thus contributing to a more effective reduction in the burden of disease associated with the virus.

Humans↗

PubMed2026

VisPan: real-time visualisation of multiplex amplicon-based sequencing panels for rapid syndromic surveillance and pathogen detection.

MOTIVATION: Infectious diseases persist as a major global public health challenge. Diverse factors, including climate change, globalization, deforestation, human-animal interactions, lifestyle choices, and various biological factors, can contribute to their emergence and reemergence. Rapid detection and characterization of (re)emerging pathogens are therefore critical for effective outbreak management and for enhancing our understanding of epidemics by monitoring the transmission, spread, evolution, and genomics of pathogens. In this context, next-generation sequencing technologies (NGS), particularly long-read platforms such as Oxford Nanopore Technologies (ONT), have opened new avenues for real-time pathogen monitoring. However, the bioinformatics bottleneck remains a challenge, emphasizing the need for efficient, accessible, and user-friendly analysis tools. RESULTS: Here, we present a tool adapted from the RAMPART software that enables real-time data visualisation of multiplex PCR syndromic panels combined with Oxford Nanopore sequencing. This real-time analysis enables rapid pathogen detection, from raw data acquisition to taxonomic assignment, within minutes. The interface offers dynamic visual tracking of the sequencing run and amplicon coverage, facilitating immediate insights during diagnostic workflows. Validation experiments confirmed the system's reliability, accurately identifying all pathogens present in complex clinical or environmental samples. This tool provides an integrated, user-friendly solution for genomic pathogen surveillance in field or clinical settings.

Software↗

PubMed2026

Whole genome sequencing of unusual Hepatitis C virus subtypes and drug resistance analysis during direct-acting antiviral therapy in India.

INTRODUCTION AND OBJECTIVES: Pangenotypic direct-acting antivirals (DAA) are effective against highly prevalent Hepatitis C virus (HCV) subtypes, but have been clinically validated almost exclusively in high-income countries. Unusual HCV subtypes may carry natural polymorphisms, potentially impacting DAA susceptibility. We conducted full-genome characterization and resistance analysis of unusual HCV subtypes in patients receiving DAA treatment. PATIENTS AND METHODS: In this prospective hospital-based study, eligible patients were screened for anti-HCV antibodies and active infection was confirmed by diagnostic 5'NCR-based HCV RNA detection. Genotyping was performed by core region sequencing, and viral load quantified by real-time PCR. For whole genome sequencing, multiplex primers were designed using alignments of global reference sequences. Sequencing was carried out using the Oxford Nanopore Technology platform. Phylogenetic analysis used multiple sequence alignment and the HCV-GLUE resource for resistance-associated substitution (RAS) analysis. RESULTS: Predominant genotype was genotype 3 in 64.3% (n = 45); genotype 6 in 21.4% (n = 15); and genotype 1 in 14.2% (n = 10). Unusual HCV subtype 6xa was detected in two patients and showed no NS5A resistance mutations. One genotype 3b patient relapsed at 24 weeks post-DAA treatment completion and carried NS5A resistance-associated substitutions 30 K and 31 M both at baseline and at relapse, conferring high-level resistance to NS5A inhibitors. CONCLUSION: This is the first report from India of whole genome sequencing of HCV subtype 6xa. The identification of NS5A resistance mutations in the 3b relapse case underscores challenges for global HCV elimination strategies.

Humans↗

PubMed2026

Alternative quadruplex real-time PCR reactions for detection and discrimination of Streptococcus pneumoniae serotypes within serogroup 6.

UNLABELLED: Streptococcus pneumoniae causes significant morbidity and mortality worldwide, and serotyping is important to assess the burden of disease that is vaccine preventable. For serotyping, the Centers for Disease Control and Prevention (CDC) use a series of 12 real-time multiplex PCRs (rmPCRs) performed in quadruplex reactions; however, rmPCR reaction 5 (rmPCR-5) for serotypes 6A, 6B, 6C, and 6D often failed at low DNA concentrations. This study investigated the cause of rmPCR-5 failure and provided alternative rmPCRs to resolve this issue. Quadruplex rmPCR target sequences were compared to S. pneumoniae reference genomes. Reactions rmPCR-5 [6ABCD, 6AB, 6BD, and 6CD] and rm-PCR-11 [37, 10F, 11BC, and 18CFBA] were compared to alternative reactions rmPCR-A1 [6ABCD, 10F, 11BC, and 18CFBA] and rmPCR-A2 [37, 6AB, 6BD, and 6CD]. All rmPCRs were tested using 10-fold serial dilutions of DNA from representative serotypes, and analytical specificity was assessed using DNA from other S. pneumoniae serotypes or various streptococci and Gram-positive cocci. Failure of rmPCR-5 was associated with overlapping 6ABCD and 6BD targets. Separation of these targets in the alternative rmPCRs-A1 and rmPCR-A2 allowed sensitive and specific detection and discrimination of serotypes 6A, 6B, 6C, and 6D, without impacting the detection of serotypes 10F, 11BC, 18CFBA, and 37. This study highlights the importance of rigorous author and peer-review to avoid manuscript errors and unintended consequences. By explaining what caused rmPCR-5 failure and proposing alternative reactions rmPCRs-A1 and rmPCR-A2, this study demonstrates the value of scientific collaboration to ensure molecular assays best serve the scientific community. IMPORTANCE: Streptococcus pneumoniae is a bacterium that can cause life-threatening infections like pneumonia and meningitis, leading to millions of deaths worldwide each year. A key feature enabling S. pneumoniae to cause disease is its sugar coating, allowing it to avoid the immune system. These surface sugars are the target of S. pneumoniae vaccines. However, vaccines only protect against some sugars and understanding which ones are on the surface of S. pneumoniae is called "serotyping." The Centers for Disease Control and Prevention (CDC) have protocols that allow us to predict S. pneumoniae serotypes by looking at its DNA. We found errors in the CDC protocols and provided a simple solution to fix them. Ultimately, having accurate serotyping protocols allows us to know how much disease is preventable by vaccine, allows us to monitor how well vaccine are working, and helps develop new vaccines if needed.

Streptococcus pneumoniae↗

PubMed2026

Near-Whole-Genome Sequencing of Peste Des Petits Ruminants Virus Lineage IV From the Savannah District, Northern Côte d'Ivoire in 2023.

Peste des petits ruminants (PPR) is a highly contagious viral disease affecting sheep and goats, causing substantial economic losses in endemic countries. In the Savannah district of Côte d'Ivoire, knowledge of the genetic diversity and molecular epidemiology of the PPR virus (PPRV) remains limited. This study investigated the genetic diversity and phylogenetic relationships of PPRV circulating in this region using whole-genome sequencing (WGS). A cross-sectional survey was conducted between September and December 2023. Nasal swabs collected from sheep and goats were screened for PPRV ribonucleic acid (RNA) using real-time reverse transcription polymerase chain reaction (RT-qPCR). Samples with low quantification cycle (Cq) values of less than 35 and successful multiplex PCR amplification profiles were selected for sequencing using the Oxford Nanopore MinION platform. Near-complete consensus genomes were generated through reference-based assembly and analysed alongside representative strains from all recognised PPRV lineages. Of the 355 samples analysed, 25 (7.0%) tested positive for PPRV RNA, with positive detections in all three surveyed regions (Poro, Tchologo and Bagoué). The four samples with the lowest Cq values, originating from all three administrative regions, were successfully sequenced, generating genomes that covered 82.0%-86.2% of the reference genome at a depth of ≥ 10 ×. The missing regions were mainly located at the 5' and 3' genomic termini, as well as in limited internal regions associated with amplicon dropout. Phylogenetic analysis revealed that all four sequences belonged to lineage IV and exhibited high nucleotide similarity (98.1%-99.9%). The Ivorian strains clustered with recent lineage IV viruses from West, North and Central Africa, whereas historical Ivorian lineages I and II formed distinct clades. These findings confirm the predominance of lineage IV in northern Côte d'Ivoire and provide baseline genomic data to support molecular epidemiological surveillance in the region.

PPRV↗

PubMed2026

Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.

The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.

Solanum tuberosum↗

PubMed2018

Quantitation of DNA Methylation by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) Assay.

The defining feature of the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) method to sensitively quantify DNA methylation is the two-step PCR approach for a multiplexed analysis of a panel of up to 12 genes in clinical samples with minimal quantities of DNA. In the first step, for up to 12 genes tested, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction. This methylation-independent amplification step produces amplicons of up to 109 copies per μL after 36 cycles of PCR. In the second step, the amplicons of the first reaction (STEP 1) are quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC). One methylated copy is detectable in 100,000 reference gene copies. Methylation is reported on a continuous scale. For the gene panel, the highest level of normal DNA methylation above which a sample would be called positive is derived by using Receiver Operating Characteristic (ROC), maximizing assay specificity and sensitivity to distinguish between normal/benign versus tumor DNA. QM-MSP can be applied to clinical samples of fresh or fixed ductal cells, ductal fluid, nipple fluid, fine needle aspirates, core biopsies, and tumor tissue sections.

Breast Neoplasms↗

PubMed2026

Simultaneous detection of glyphosate and glufosinate target-site resistance in Eleusine indica via multiplex TaqMan qPCR.

BACKGROUND: Continuous use of glyphosate followed by glufosinate-ammonium has selected for multiple resistance to both herbicides in Eleusine indica worldwide. Managing such resistant weeds requires fast, accurate molecular detection assay. To address this critical need, we developed a robust multiplex TaqMan quantitative (q)PCR assay that simultaneously detects five well-characterized target-site resistance markers in E. indica: EPSPS copy number variation; T102I in EPSPS; P106A and P106S in EPSPS; and S59G in GS1-1. RESULTS: The multiplex qPCR assay showed analytical specificity when tested on genomic DNA from nine reference accessions: three susceptible, three glyphosate-resistant (with EPSPS CNV) and three multiple-resistant. Subsequent analysis of 56 field-collected samples demonstrated 98.2% concordance (55 of 56) with Sanger sequencing across all five resistance-associated markers: EPSPS CNV, T102I, P106A, P106S and GS1-1 S59G, confirming the reliability and practical value of the multiplex qPCR assay. Only samples 7-8 showed discordance at EPSPS position 102, where Sanger chromatograms showed overlapping peaks at this position, which is likely to be a result of heterozygous mutation distribution among amplified EPSPS gene copies. This case further underscores the advantages of the multiplex qPCR assay over Sanger sequencing in detection sensitivity and accuracy. Moreover, a strong correlation (R2 = 0.8935) in gene copy number estimation between the two methods across all samples further supports the reliability of the qPCR assay. CONCLUSIONS: In summary, this study delivers a simple, robust and high-throughput diagnostic tool for the rapid, simultaneous identification of dual herbicide target-site resistance in goosegrass, offering superior sensitivity, quantitative resolution and throughput compared with Sanger sequencing. © 2026 Society of Chemical Industry.

Herbicides↗