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PubMed2026

Diagnostic performance of panfungal PCR on tissue specimens for the diagnosis of invasive fungal diseases: a systematic review and meta-analysis of the Fungal PCR Initiative (FPCRI).

UNLABELLED: Invasive fungal diseases are difficult to diagnose because of the limited sensitivity of culture. Panfungal PCR amplicon sequencing assays (targeting ribosomal RNA, such as 18S, 28S, ITS) are recommended for fungal identification in histopathology samples showing fungal elements. However, data describing its overall performance and consistency are lacking. This systematic literature review and meta-analysis assessed the performance of panfungal PCR on formalin-fixed paraffin-embedded (FFPE) and non-fixed (fresh or frozen) tissue samples. A systematic literature search was performed to include studies reporting the use of panfungal PCR for fungal identification in FFPE or non-fixed tissue samples. PCR sensitivity and specificity were assessed using the reference standard of histopathology showing fungal elements. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Pooled estimates were obtained using random-effects meta-analysis. Twenty-eight studies were included. In FFPE samples (18 studies, 852 samples), sensitivity and specificity were 75.4% (95% confidence interval [CI], 59.2-86.6) and 93.5% (70.2-98.9), respectively. Sensitivity in non-fixed samples (13 studies, 207 samples) was 86.5% (74.7-93.3), while specificity could not be assessed (insufficient data). Comparative analyses showed a significantly higher sensitivity of panfungal PCR over culture (88.2%; 76-94.7 vs 52.2%; 39-65, P = 0.001). Sub-analyses could not demonstrate the superiority of one PCR target over another due to limited data. Panfungal PCR exhibited adequate sensitivity and good specificity in FFPE samples. Sensitivity was even higher in non-fixed samples and largely superior to culture. Nevertheless, large interstudy variability was observed, warranting interlaboratory studies to define the optimal PCR target and standardized protocols. IMPORTANCE: Invasive fungal diseases are difficult to diagnose because of the low sensitivity of culture. Panfungal PCRs are widely used for fungal identification in tissue specimens but suffer from heterogeneous procedures and performance. This meta-analysis shows an acceptable sensitivity (75.4% and 86.5% in fixed and non-fixed samples, respectively) and good specificity (93.5%) of panfungal PCR, supporting its use, not only on histopathology-positive fixed samples but also in non-fixed samples concomitantly with other diagnostic tools (cultures and fungal-specific PCRs if available). These results provide a strong basis for further standardization of panfungal PCR techniques via interlaboratory assays to assess reproducibility and optimize analytical protocols. CLINICAL TRIALS: This study is registered with PROSPERO as CRD42023461148.

Humans↗

PubMed2025

Viral viability markers of SARS-CoV-2: a comparison of cell culture, genomic RNA RT-PCR, and subgenomic RNA RT-PCR.

UNLABELLED: Accurate methods to assess viral viability are crucial for determining isolation duration and antiviral therapy in immunocompromised patients. Although cell culture (CC) is the gold standard, it has limitations. Cycle threshold (Ct) values from genomic RNA (gRNA) RT-PCR and subgenomic RNA (sgRNA) RT-PCR have been proposed as markers of active viral replication. This study evaluated the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. This study aimed to evaluate the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. We conducted a prospective study on immunocompromised patients with suspected SARS-CoV-2 infection at a tertiary hospital (May 2021 to May 2023). Nasopharyngeal swabs were inoculated into Vero E6 cells and tested for gRNA using RT-PCR (Cobas 6800, Roche) and for sgRNA (non-commercial RT-PCR). The sensitivity (S), specificity (SP), positive (PPV) and negative predictive value (NPV), and accuracy were calculated using CC as the gold standard. Among 285 samples from 108 patients, gRNA RT-PCR had high S and NPV (1.0) but low SP (0.24) and moderate PPV (0.63). Ct analysis improved performance in extreme but not intermediate values. A Ct ≤ 30 maximized S but had low SP; Ct ≤ 25 yielded S (0.88), SP (0.89), PPV (0.92), NPV (0.84), and accuracy (0.88); sgRNA showed the highest S (0.99), SP (0.96), PPV (0.97), NPV (0.99), and accuracy (0.98). sgRNA detection is the best marker for identifying viable SARS-CoV-2, aiding decisions on isolation, antiviral treatment, or delaying chemotherapy in immunocompromised patients. IMPORTANCE: Identifying whether a patient still has contagious SARS-CoV-2 is essential for managing isolation, antiviral treatment, and other clinical decisions-especially in immunocompromised individuals. While viral culture is the gold standard for confirming viral viability, it is slow, expensive, and not widely available. Many hospitals rely on RT-PCR tests, but these detect viral genetic material whether or not the virus is still active. This study shows that detecting subgenomic RNA (sgRNA), a molecule only present when the virus is actively replicating, is a highly accurate way, a molecule only present when the virus is actively replicating, is a highly accurate way to determine whether the virus is still viable. Compared to standard PCR or viral culture, sgRNA testing better predicts who is truly infectious. These findings support sgRNA as a useful tool to guide clinical management and infection control in vulnerable patients.

Humans↗

PubMed2025

Performance of Subgenomic RT-PCR for Predicting SARS-CoV-2 Infectivity Compared to Genomic RT-PCR and Culture Isolation.

SARS-CoV-2 clinical samples can be detected as positive for a long period of time using real-time RT-PCR, even when patients are no longer infectious. Viral culture is the gold standard for assessing a patient's infectivity, but it is a time-consuming technique and lacks sensitivity. SARS-CoV-2 subgenomic RNA (sgRNA) detection has been used as a proxy for assessing the infectivity but only a limited number of studies have described its use in vitro and in clinical samples. This study aimed to evaluate the correlation between results from viral culture, genomic RT-PCR (gRT-PCR), and subgenomic RT-PCR (sgRT-PCR) during in vitro infection and in clinical samples. In vitro viral replication kinetics showed that both genomic RNA (gRNA) and subgenomic RNA (sgRNA) levels remained stable up to 21 days in the absence of replication-competent virus. Using clinical samples, sgRNA was detected in 87.5% of culture-positive samples, demonstrating better performances than gRT-PCR (Positive predictive value (PPV) 93.3% and Negative predictive value (NPV) of 87.5%) and an almost perfect agreement with culture results (Cohen κ = 0.81 [95% CI: 0.66-0.95]). These findings suggest that testing for sgRNA and/or using a gRNA Ct cut-off of 21.2 could be used as a proxy to determine the presence of SARS-CoV-2 replication-competent virus.

Humans↗

PubMed2026

Sharkmer: repurposing PCR primers for targeted genome assembly using in silico PCR.

SUMMARY: We introduce an in silico PCR (sPCR) method for the assembly of specific genomic regions spanned by PCR primers using raw sequence reads. This allows a user to quickly isolate the exact regions that are abundant in public archives of gene sequences, leveraging the decades of work that have gone into optimizing primer sequences for benchtop PCR. We implement sPCR in sharkmer as a targeted de Bruijn graph assembler seeded with the forward primer sequence and terminated with the reverse primer sequence. This is useful for a variety of routine tasks, including validating the species identity of a dataset, identifying contaminants, and quickly building phylogenies from raw sequence data. AVAILABILITY AND IMPLEMENTATION: sharkmer is written in Rust. Code, instructions for installation and use, tests, and other resources are available in the GitHub repository at https://github.com/caseywdunn/sharkmer and at Zenodo with DOI 10.5281/zenodo.19020708. It can also be installed via bioconda.

Software↗

PubMed2026

Clinical impact of 16S rRNA RC-PCR NGS on infectious disease management.

16S rRNA metagenomics provides a culture-independent method for diagnosing infections with fastidious or uncultivable organisms, guiding targeted therapy, and detecting polymicrobial communities. This study utilizes reverse complement (RC)-PCR next-generation sequencing (NGS) to accurately identify bacterial pathogens from clinical specimens and assess its impact on clinical decision-making, setting it apart from conventional 16S sequencing approaches. A retrospective analysis of an ISO 15189 accredited 16S RC-PCR NGS diagnostic workflow targeting the V1-6 and V9 regions of the 16S rRNA gene was conducted over a 2-year period, including 390 clinical specimens from 316 patients. 16S RC-PCR NGS results were discussed in a multidisciplinary consultation and subsequently reported to the clinic. In total, 1,283 RC-PCR results were analyzed, of which 517 were from clinical specimens, 284 were negative controls, 66 were positive controls, and 416 were from wet lab and bioinformatic pipeline validation. 16S RC-PCR NGS assay detected bacterial taxa in 179/390 (45.9%) of clinical specimens, while 201/390 (51.5%) were negative, and 10/390 (2.6%) yielded uninterpretable results. The specimen types pus, pleural fluid, and heart valves exhibited the highest positivity rate (68% to 70%). Overall, 16S RC-PCR NGS influenced diagnostic decision making in 145/282 (51.4%) clinical cases and guided therapeutic management in 77/282 (27.3%) cases. Results providing definite evidence for either the presence or absence of bacterial infection were considered clinically valuable. Integration of 16S RC-PCR NGS pathogen detection with multidisciplinary consultation markedly improved clinical management, directly impacting diagnosis and treatment of complex clinical cases in a tertiary care setting. The effect was most pronounced in brain abscess patients, where RC-PCR results guided treatment decisions in 9/13 (69.2%) of cases.IMPORTANCETimely and accurate diagnosis is essential for managing serious infections, yet clinicians often face situations where routine laboratory tests do not provide clear answers. This study demonstrates that next-generation sequencing (NGS) of the bacterial 16S rRNA gene can decisively resolve these uncertainties. By revealing whether bacteria are present in clinical specimens, this approach influenced clinical reasoning and supported treatment decisions across a variety of challenging cases. 16S reverse-complement PCR was especially powerful for brain abscesses and infections where the causative microorganism was unclear, providing clarity that directly improved patient care. These findings show that integrating advanced sequencing with expert clinical interpretation can enhance the management of complex infections and support more confident, evidence-based therapy.

Humans↗

PubMed2026

Clinical performance of the Abbott RealTime Mycobacterium tuberculosis (MTB) PCR on bronchoscopic specimens for diagnosing pulmonary tuberculosis.

PURPOSE: We evaluated the performance of the Abbott RealTime Mycobacterium tuberculosis (MTB) PCR (RT MTB) on bronchoscopic specimens using conventional culture as the reference standard in a low-Tuberculosis (TB) prevalence setting. METHODS: A total of 6,988 specimens (4,682 bronchial aspirates [BAS] and 2,306 bronchoalveolar lavages [BAL]) from 4,118 patients with suspected pulmonary TB were included. When BAS and BAL specimens from the same bronchoscopy procedure were available, these were mixed 1:1 prior to culture inoculation and PCR testing. Following processing, specimens were inoculated into a Löwenstein-Jensen and a Bactec MGIT 960 tube and incubated at 37 °C for 3 months and at 35 °C for 8 weeks, respectively. RT MTB was performed as indicated by the manufacturer. RT MTB targets the insertion sequence IS6110 and the protein antigen B (PAB) gene, both highly conserved within the Mycobacterium tuberculosis complex. Whole genome Next generation sequencing of clinical MTBC isolates was performed when indicated. RESULTS: Among the 104 culture-positive specimens, 84 (1.2%) were detected by PCR. Additionally, 16 specimens (0.3% of all samples), from 16 patients, were PCR-positive despite negative culture results. Conversely, 20 specimens (0.4% of all samples), from 19 patients, were culture-positive but not detected by PCR. No significant differences were found between PCR-positive and PCR-negative specimens with respect to the number of IS6110 copies per isolate or PAB gene sequences (P = 0.69). Finally, there were 4,683 specimens (97.4%) from the remaining 4,014 patients tested PCR-negative/Culture-negative. Following the resolution of discrepancies based on clinical grounds the sensitivity and specificity of RT MTB were 83.9% (CI 95%, 76.0-90.0) and 99.9% (CI 95%, 99.9-99.9), respectively. These results exceed the minimum performance requirements defined in the WHO Target Product Profiles for molecular TB diagnostics. Although RT MTB is not a point-of-care test but rather a moderate-complexity automated NAAT, it is recommended by WHO as part of the Abbott RealTime MTB/MTB RIF-INH testing algorithm, in which MTBC detection by RT MTB is followed by reflex testing with the MTB RIF/INH assay for detection of rifampicin and isoniazid resistance. CONCLUSION: RT MTB shows a good performance on bronchoscopic specimens.

Mycobacterium tuberculosis↗

PubMed2025

Correlation Between Infectivity and qRT-PCR Values for Murine Norovirus Recovered from Frozen Berries.

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis globally, with frozen berries frequently implicated in foodborne outbreaks. Current surveillance relies on quantitative reverse transcription PCR (qRT-PCR), which cannot differentiate between infectious and non-infectious viral particles, complicating risk assessment. This study is aimed to establish the minimum viral load on frozen berries detectable by qRT-PCR that corresponds to infectious virus, using murine norovirus (MNV) as a surrogate for HuNoV. Frozen raspberries were artificially inoculated with serial dilutions of MNV (7.1-1.0 log PFU/25 g) and processed using the ISO 15216:2017 method. Infectious virus was quantified by plaque assay, and viral RNA was detected by qRT-PCR. The limit of detection (LOD) for cell culture was 3.1 log PFU/25 g, whereas qRT-PCR extended sensitivity to 1.0 log PFU/25 g (Ct value at 36.7 ± 0.6), representing a 2-log difference. Recovery rates for infectious virus exceeded the ISO 15,216 minimum threshold (1%), and PCR inhibition was negligible. We next examined the extraction efficiency for both infectious MNV and its genetic material from frozen strawberries at inoculation levels higher than the LOD, and observed that the viral recovery from frozen strawberries is very similar to viral recovery from frozen raspberries with no significant differences between them. The disparity between LODs indicates that a substantial proportion of MNV genomes detected by qRT-PCR do not represent infectious particles, aligning with previous findings that one PFU may correspond to multiple genome copies. Given that many surveillance studies report high Ct values (> 35), our data suggest that such detections may not indicate viable virus, underscoring the importance of contextualizing qRT-PCR results with epidemiological evidence. These findings highlight the need for cautious interpretation of surveillance data, particularly for public health decision-making.

Norovirus↗

PubMed2026

PCR analysis of insertion sequences leads to the generation of artefact amplicons.

Insertion sequences (ISs) are small, self-mobilizing DNA elements widespread across prokaryotic genomes, including chromosomes and plasmids. IS elements frequently co-localize with antimicrobial resistance (AMR) genes and mediate their mobilization, often as part of larger genomic structures that encompass multiple IS elements and antibiotic resistance genes. In this study, we employed Polymerase Chain Reaction (PCR) to amplify DNA sequences containing two copies of an IS26 element from two Escherichia coli ST131 isolates. While the respective PCRs generated products of the expected size, we also observed multiple amplicons of unexpected sizes, which could be misinterpreted as population heterogeneity attributed to IS mobilization. By extracting, re-amplifying and sequencing individual PCR products, we demonstrate that these amplicons of unexpected sizes were indeed artefact products generated during the PCR reaction, likely mediated by within-PCR recombination of the IS26 sequences. Furthermore, PCRs with equally oriented primers, each located close to an IS26 element, also generated artefact amplicons. This research highlights the limitations of using PCR to assess DNA sequences encoding multiple copies of an IS element and therefore, the presence of these genomic structures or the mobilization of the respective IS elements should not be assessed by diagnostic PCR alone but be corroborated with complementary techniques.

ESBL↗

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

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗

PubMed2026

A 29-plex MOL-PCR assay for simultaneous detection of selected major, non-typing, and accessory virulence genes in Clostridium perfringens.

Clostridium perfringens is an important pathogen of humans and animals, responsible for a broad spectrum of diseases mediated by diverse toxins and virulence factors. Precise and extended toxin-gene profiling is valuable for strain characterization and molecular epidemiological surveillance. Here, we describe the development of a 29-plex Multiple Oligonucleotide Ligation PCR (MOL-PCR) assay that enables the simultaneous detection of a large and important panel of 27 C. perfringens toxin-related genes - covering major typing toxins as well as an extended panel of non-typing and accessory virulence genes - thus moving beyond the classical toxinotyping framework. The assay was evaluated in comparison with six multiplex qPCR assays. In both systems, the gene encoding the Clostridium perfringens-specific serine O-acetyltransferase (EpsC) was used as a molecular marker for species confirmation, and an internal amplification control was included to detect potentially false-negative results. Analytical specificity testing confirmed exclusive amplification in C. perfringens and sequencing confirmed the toxin-gene profiles of reference strains. Comparative analysis of 72 reference and field isolates (1,944 data points) demonstrated complete concordance for 637 positive detections, yielding 100% positive agreement and 99.7% negative agreement relative to the comparative qPCR method. The limit of detection was 100 fg/µl (approx. 3 × 101 genome equivalents; GE) for qPCR and 1  pg/µl (approx. 3 × 102 GE) for MOL-PCR. Despite its high multiplex level, MOL-PCR showed high agreement with qPCR. The developed MOL-PCR method provides a rapid, high-throughput, and cost-effective tool for expanded toxin-gene profiling of C. perfringens isolates targeting major typing toxins and selected non-typing and accessory virulence genes. Therefore, it may support advanced toxin-gene characterization, molecular epidemiology, and One Health-oriented surveillance of evolving virulence landscapes.

Clostridium perfringens↗

PubMed2026

First report of tomato spotted wilt virus (Orthotospovirus tomatomaculae) and phytoplasma in China aster and development of duplex PCR, LAMP, and qPCR assays for rapid detection.

UNLABELLED: China aster (Callistephus chinensis) is an economically important ornamental crop widely cultivated for cut flowers and landscaping. During field surveys conducted in three districts of Karnataka, India, China aster plants exhibiting chlorotic and necrotic ring spots, leaf deformation, and witches' broom symptoms were collected and analyzed to determine the causal agents. Mechanical inoculation of symptomatic leaf sap onto cowpea (Vigna unguiculata cv. C-152) produced characteristic chlorotic and necrotic ring spots on newly emerging leaves indicating the presence of an infectious viral agent. Serological assay by DAC-ELISA followed by RT-PCR confirmed the presence of tomato spotted wilt virus (TSWV, Orthotospovirus tomatomaculae) in symptomatic plants. Similarly the plants exhibiting witches' broom symptoms tested positive for phytoplasma infection using universal and Nested primers PCR assays targeting the 16S rRNA gene. Sequence analysis of TSWV CP gene revealed more than 97% nucleotide identity with TSWV isolates reported from India and other countries. Based on these results, one representative isolate was selected for complete genome sequencing. The complete sequences of the L, M, S RNA segements were amplified cloned, and sequenced showing more than 97% nucleotide identity with global TSWV isolates available in database. Sequence analysis of 16S rRNA gene of the phytoplasma associated with witches' broom symptoms was identified as 'Candidatus Phytoplasma australasiaticum' belonging to the 16SrII-D subgroup, sharing 99.2% nucleotide identity with previously reported isolates. Phylogenetic analysis further supported the placement of both the TSWV and phytoplasma isolates within their respective taxonomic groups. To facilitate rapid and sensitive diagnosis, quantitative PCR (qPCR) and RT-LAMP assays were developed for TSWV detection. In addition a duplex PCR assay was optimized for simultaneous detection of TSWV and phytoplasma from infected China aster plants in a single reaction. This study represents the first reports of the complete genome characterization of TSWV and phytoplasma infection in China aster in India along with the development of sensitive qPCR, RT-LAMP, and duplex PCR assays for rapid detection of these pathogens providing valuable tools for disease diagnosis, epidemiological studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05038-w.

China aster↗

PubMed2022

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics.

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Humans↗

PubMed2025

Multiplex PCR assay for the rapid detection of Klebsiella pneumoniae pathotypes.

Introduction. Klebsiella pneumoniae (Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence.Gap Statement. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision.Aim. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance.Methodology. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (rmpA, rmpA2, iucA, peg344 and iroB), carbapenem-resistant (bla NDM, bla OXA-48-like and bla KPC) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity.Results. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl-1. Additionally, validation through sequencing confirmed the accuracy of detected amplicons.Conclusion. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.

Klebsiella pneumoniae↗

PubMed2026

Clinical performance of the urine-based TERT promoter AbsoluteQ Digital PCR for non-invasive detection of bladder cancer.

Bladder cancer (BC) is the ninth most common cancer worldwide, with urothelial carcinoma accounting for approximately 90% of all cases and presenting predominantly as non-muscle-invasive disease. Due to its high recurrence rate and the need for long-term surveillance, BC is associated with the highest lifetime treatment costs per patient among all cancers, making its effective management a significant clinical and economic challenge. The most frequently identified variants in the TERT gene promoter are c.-124C>T (C228T) and c.-146C>T (C250T), located within a region characterized by high guanine-cytosine (GC) content, which makes amplification challenging. We aimed to validate the AbsoluteQ Digital PCR assay for the detection of urine-based TERT promoter variants for the diagnosis of urothelial bladder cancer and to assess its diagnostic performance in comparison with standard methods. Urine samples were collected from patients with histopathologically confirmed bladder cancer (n = 58) and compared with a control group (n = 55). The C228T and C250T variants were tested using the AbsoluteQ Digital PCR assay. Sensitivity, specificity, and predictive values were calculated to evaluate the performance of the assessed method. The AbsoluteQ Digital PCR demonstrated superior diagnostic performance compared to conventional Sanger sequencing for detecting TERT promoter variants, achieving a sensitivity of 89.65% (95% CI: 78.16-95.72) and a specificity of 100% (95% CI: 91.87-100), with no false positives observed. Given its robustness and clinical relevance, AbsoluteQ Digital PCR is emerging as a promising tool for non-invasive molecular diagnostics targeting TERT promoter variants.

Humans↗

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↗

PubMed2026

Development and optimization of T-ARMS PCR assays for detection of lethal haplotypes of TADA2A, UR1B, and PORL1B in pigs in Vietnam.

Marker-assisted selection has increasingly relied on single-nucleotide polymorphisms (SNPs) as robust genetic markers, particularly in livestock breeding programs. In pig farming, embryonic mortality significantly affects litter size, and SNPs in reference genes have been implicated as potential causal factors. We developed and optimized a tetra-primer amplification refractory mutation system (T-ARMS) PCR assay for rapid, cost-effective detection of SNPs in 3 candidate genes-TADA2A, PORL1B, URB1-that are associated with embryonic lethality and reproductive performance. Primer sets were designed based on known mutation sites and validated using synthetic gene constructs and porcine genomic DNA from pigs of Duroc and Landrace breeds. Optimization of annealing temperatures and primer concentration ratios yielded distinct and reproducible allele-specific amplicon patterns that were corroborated by PCR-RFLP and Sanger sequencing. Our T-ARMS PCR protocol, which requires minimal equipment and reduces processing time to <3 h, had high specificity and efficiency in differentiating wild-type, heterozygous, and homozygous mutant genotypes in 20 Duroc and 20 Landrace pigs. Our Tetra-ARMS PCR assay is a robust and economically viable tool for SNP genotyping in pig breeding programs, potentially contributing to the reduction of embryonic lethality and the improvement of overall reproductive outcomes.

Sus scrofa↗

PubMed2025

Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions.

Mycobacterium orygis, a recently defined member species of Mycobacterium tubercuolsis complex (MTBC), is emerging as a major threat to zoonotic tuberculosis control, especially in the Asian Subcontinent. The dearth of low-cost diagnostic assay to differentiate M. orygis from other members of the MTBC leads to unavailability of information about the actual burden of this species in human and animal population. In this study, we developed a multiplex PCR for distinguishing M. orygis from other MTBC based on two M. orygis-specific nonsynonymous point mutations in mbtG and fadD23 genes identified by comparative genome analysis. The specificity of the assay shows that a 434 bp IS1081 fragment was amplified from common MTBC species including M. orygis while 240 bp and 181 bp mbtG and fadD23 gene fragments were amplified only from M. orygis. No amplification was observed for nontuberculous Mycobacterium (NTM) and non-Mycobacterial pathogens. The multiplex PCR assay showed a detection limit of 32 pg of M. orygis DNA. Furthermore, a total of 85 tuberculous-like lesions in the different tissues of slaughtered cattle were tested for identification of the M. orygis, and the results showed IS1081, mbtG and fadD23 amplicons in three tissue DNA extracts confirming they contain M. orygis DNA. Also, a single IS1081 amplicon was amplified from one tissue sample signifying presence of DNA of any MTBC species other than M. orygis. An established TaqMan real time PCR assay targeting region of differences (RD) in M. orygis genome was carried out to validate the result of the assay. This showed 100 % accuracy of the in-house developed multiplex PCR.

Mycobacterium orygis↗