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Development and application of recombinase polymerase amplification assay for rapid detection of sp.Parasitology Nov 2023sp. is a common parasite in the intestinal tract of humans and animals. The clinical diagnosis of sp. mainly depends on the microscopic observation of parasite, which...
sp. is a common parasite in the intestinal tract of humans and animals. The clinical diagnosis of sp. mainly depends on the microscopic observation of parasite, which can lead to false-negative results. An accurate and convenient diagnostic approach for sp. infection is crucial for effectively preventing and controlling blastocystosis. Herein, we developed a recombinase polymerase amplification (RPA) method for detecting sp. The results showed that the DNA amplification by RPA established in this study could be performed within 5 min at 37°C, with maximum band intensity observed at 30 min. The minimum detection limit of RPA was 100 fg L, consistent with conventional polymerase chain reaction (cPCR). Furthermore, the RPA method exhibited no cross-reactivity with 7 other non-target pathogens in the intestinal tract. Next, the newly established RPA method was used to analyse 40 fecal samples collected clinically, and the detection results were consistent with cPCR. These results corroborate that the newly developed RPA method has good sensitivity and specificity and offers the advantage of short detection times, which can be harnessed for differential diagnosis and rapid detection of sp.
Topics: Humans; Animals; Recombinases; Blastocystis; Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Blastocystis Infections; Real-Time Polymerase Chain Reaction
PubMed: 37860882
DOI: 10.1017/S0031182023000975 -
Journal of Food Protection Aug 2023Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them...
Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them targeting the amplification of two regions of the insect's mitochondrial cytochrome c oxidase subunit I (COI-Fa and COI-Fb) and the other targeting a region of the nuclear protein-coding wingless (wg) gene. Using singleplex and multiplex polymerase chain reaction (PCR), we evaluated the three sets of primers using genomic DNA (gDNA) from 48 insect species including food storage insect pests and known vectors of foodborne pathogens. Seven plant-based food matrices were also evaluated for exclusivity testing. Additionally, we spiked fragments from five insect species in a selected food matrix (whole wheat flour). Singleplex and multiplex PCR amplified single specific bands (401-449 bp), corresponding to the wg gene, from insect species belonging to families Blattidae and Formicidae, and in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The COI-Fa primers amplified specific bands (171-188 bp) in all Dipteran species and the COI-Fb primers amplified a specific band (∼140 bp) in DNA from Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and P. interpunctella. However, the presence of specific bands in most Coleopterans was not consistent. No amplicon bands were observed in any of the food matrixes tested and the expected pattern of amplicon bands was seen in multiplex reactions using gDNA from spiked food samples. Our multiplex PCR assay targeted specific groups of insects that commonly contaminate foods without amplifying bands from the food matrixes tested; thus, molecular methods may be suitable for detecting insects or their fragments in foods.
Topics: Humans; Animals; Multiplex Polymerase Chain Reaction; Flour; Triticum; Insecta; DNA; Coleoptera; DNA Primers; Allergens
PubMed: 37348561
DOI: 10.1016/j.jfp.2023.100120 -
Journal of Infection and Chemotherapy :... Aug 2023For patients with coronavirus disease 2019 (COVID-19) requiring hospitalization, extending isolation is warranted. As a cautious protocol, ending isolation based on...
Judicious ending of isolation based on reverse transcription-polymerase chain reaction (RT-PCR) cycle threshold only for patients with coronavirus disease 2019 (COVID-19) requiring in-hospital therapy for longer than 20 days after symptom onset.
BACKGROUND
For patients with coronavirus disease 2019 (COVID-19) requiring hospitalization, extending isolation is warranted. As a cautious protocol, ending isolation based on polymerase chain reaction cycle threshold (Ct) value was introduced for patients requiring therapy for >20 days after symptom onset.
METHOD
We compared a Ct-based strategy using Smart Gene® between March 2022 and January 2023 with a preceding control period (March 2021 to February 2022) when two consecutive negative reverse transcription-polymerase chain reaction tests using FilmArray® were required for ending isolation. Ct was evaluated on day 21, and ending isolation was permitted in patients with Ct ≥ 38. Although patients with Ct 35-37 were transferred to a non-COVID-19 ward, isolation was continued.
RESULTS
The duration of stay on a COVID-19 ward in the Ct group was 9.7 days shorter than that in controls. The cumulative number of tests was 3.7 in controls and 1.2 in the Ct group. There was no nosocomial transmission after ending isolation in either group. The number of days from symptom onset to testing was 20.7 ± 2.1 in Ct group, and five patients had Ct < 35, nine Ct 35-37, and 71 Ct ≥ 38. No patients were moderately or severely immunocompromised. Steroid use was an independent risk factor for prolonged low Ct (odds ratio 9.40, 95% confidence interval 2.31-38.15, p = 0.002) CONCLUSIONS: The efficacy of ending isolation based on Ct values could improve bed utilization without the risk of transmission among patients with COVID-19 requiring therapy for >20 days after symptom onset.
Topics: Humans; COVID-19; SARS-CoV-2; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Hospitals; Polymerase Chain Reaction; COVID-19 Testing
PubMed: 37209841
DOI: 10.1016/j.jiac.2023.05.007 -
Neurocritical Care Jun 2024Patients with hemorrhagic stroke and an external ventricular drain in situ are at risk for ventriculostomy-related-infections (VRI). Because of the contamination of the...
Broad Range Eubacterial Polymerase Chain Reaction of Cerebrospinal Fluid Reduces the Time to Exclusion of and Costs Associated with Ventriculostomy-Related Infection in Hemorrhagic Stroke.
BACKGROUND
Patients with hemorrhagic stroke and an external ventricular drain in situ are at risk for ventriculostomy-related-infections (VRI). Because of the contamination of the cerebrospinal fluid (CSF) with blood and the high frequency of false negative CSF culture, the diagnosis of VRI remains challenging. This study investigated the introduction of CSF broad range eubacterial polymerase chain reaction (ePCR) and its effect on frequency and duration of antibiotic therapy for VRI, neurocritical care unit (NCCU) length of stay, related costs, and outcome.
METHODS
Between 2020 and 2022, we prospectively included 193 patients admitted to the NCCU of the University Hospital of Zürich with hemorrhagic stroke and an external ventricular drain for more than 48 h. Patient characteristics, serum inflammatory markers, white blood cell count in CSF, use and duration of antibiotic treatment for VRI, microbiological findings (CSF cultures and ePCR tests), and NCCU length of stay were compared in patients with no infection, noncerebral infection, suspected VRI, and confirmed VRI. Data of patients with suspected VRI of this cohort were compared with a retrospective cohort of patients with suspected VRI treated at our NCCU before the introduction of CSF ePCR testing (2013-2019).
RESULTS
Out of 193 patients, 12 (6%) were diagnosed with a confirmed VRI, 66 (34%) with suspected VRI, 90 (47%) with a noncerebral infection, and 25 (13%) had no infection at all. Compared with the retrospective cohort of patients, the use of CSF ePCR resulted in a reduction of patients treated for suspected VRI for the whole duration of 14 days (from 51 to 11%). Furthermore, compared with the retrospective group of patients with suspected VRI (n = 67), after the introduction of CSF ePCR, patients with suspected VRI had shorter antibiotic treatment duration of almost 10 days and, hence, lower related costs with comparable outcome at 3 months.
CONCLUSIONS
The use of CSF ePCR to identify VRI resulted in shorter antibiotic treatment duration without changing the outcome, as compared with a retrospective cohort of patients with suspected VRI.
Topics: Humans; Male; Ventriculostomy; Female; Middle Aged; Aged; Hemorrhagic Stroke; Anti-Bacterial Agents; Polymerase Chain Reaction; Length of Stay; Retrospective Studies; Cerebrospinal Fluid; Prospective Studies
PubMed: 38087175
DOI: 10.1007/s12028-023-01888-x -
Forensic Science International. Genetics Jul 2024Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently...
Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)-epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process.
Topics: Humans; DNA; Cancellous Bone; Microsatellite Repeats; Femur; DNA Fingerprinting; Polymerase Chain Reaction; Male; Real-Time Polymerase Chain Reaction
PubMed: 38833778
DOI: 10.1016/j.fsigen.2024.103067 -
Molecular Aspects of Medicine Apr 2024Well-characterized reference materials support harmonization and accuracy when conducting nucleic acid-based tests (such as qPCR); digital PCR (dPCR) can measure the... (Review)
Review
Well-characterized reference materials support harmonization and accuracy when conducting nucleic acid-based tests (such as qPCR); digital PCR (dPCR) can measure the absolute concentration of a specific nucleic acid sequence in a background of non-target sequences, making it ideal for the characterization of nucleic acid-based reference materials. National Metrology Institutes are increasingly using dPCR to characterize and certify their reference materials, as it offers several advantages over indirect methods, such as UV-spectroscopy. While dPCR is gaining widespread adoption, it requires optimization and has certain limitations and considerations that users should be aware of when characterizing reference materials. This review highlights the technical considerations of dPCR, as well as its role when developing and characterizing nucleic acid-based reference materials.
Topics: Humans; Polymerase Chain Reaction; Nucleic Acids; Real-Time Polymerase Chain Reaction
PubMed: 38359699
DOI: 10.1016/j.mam.2024.101256 -
Haematologica Jun 2024Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia...
Multi-gene measurable residual disease assessed by digital polymerase chain reaction has clinical and biological utility in acute myeloid leukemia patients receiving venetoclax/azacitidine.
Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia (AML). Measurable residual disease (MRD) using flow cytometry is a valuable tool for the prediction of relapse in AML using conventional therapies and ven/aza; however, the prognostic value for broadscale molecular MRD after ven/aza treatment is less clear. We aimed to determine the utility of retrospective assessment using multi-gene molecular MRD by droplet digital polymerase chain reaction (ddPCR). We found this approach correlates with outcomes in a cohort of patients receiving frontline ven/aza for AML. The predictive value of ddPCR MRD persisted when NPM1 mutations were removed from analysis, as well as after adjustment for the impact of stem cell transplant on outcomes. Late achievement of MRD negativity, including after SCT, was still associated with superior outcomes compared to persistently detectable MRD. We further explored the impact of ven/aza on the burden of different classes of mutations, and identified the persistence of splicing factor mutations, commonly associated with MDS, as a consistent finding after ven/aza treatment. These data add to our understanding of the effects of ven/aza on AML disease biology and provide details on molecular depth of remission that can guide prospective trials in the future.
Topics: Humans; Leukemia, Myeloid, Acute; Neoplasm, Residual; Nucleophosmin; Sulfonamides; Bridged Bicyclo Compounds, Heterocyclic; Aged; Male; Female; Azacitidine; Middle Aged; Antineoplastic Combined Chemotherapy Protocols; Mutation; Polymerase Chain Reaction; Prognosis; Aged, 80 and over; Retrospective Studies; Adult; Treatment Outcome
PubMed: 38105738
DOI: 10.3324/haematol.2023.283790 -
Archives of Pathology & Laboratory... May 2024Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted...
CONTEXT
Droplet digital polymerase chain reaction (ddPCR) is a sensitive method to detect common pathogenic EGFR mutations in non-small cell lung cancer. Although targeted assays have not been specifically designed to detect them, uncommon EGFR mutations have been linked to response to targeted therapy.
OBJECTIVE
To describe atypical ddPCR patterns that correspond to uncommon but clinically actionable EGFR mutations.
DESIGN
A cohort of 1134 consecutive non-small cell lung cancers that underwent targeted next-generation sequencing was reviewed. Uncommon EGFR mutations involving probe binding sites were evaluated by ddPCR.
RESULTS
Two hundred fifty-five of 1134 cancers (22.5%) harbored pathogenic EGFR mutations. One hundred eighty-six of 255 (72.9%) had canonical EGFR exon 19 deletion or exon 21 p.L858R variants designed for detection by ddPCR. An additional 25 of 255 cases (9.8%) had uncommon EGFR mutations within the probe-binding site, including 1 case with concurrent uncommon mutations in both exon 19 and exon 21. These mutations included uncommon EGFR exon 19 deletions (n = 6), EGFR exon 19 substitutions p.L747P (n = 3) and p.L747A (n = 1), dinucleotide substitutions leading to EGFR p.L858R (n = 5), EGFR exon 21 substitutions p.K860I (n = 1) and p.L861Q (n = 9), and EGFR p.[L858R;K860I] (n = 1). Droplet digital polymerase chain reaction generated atypical but reproducible signal for each of these uncommon variants.
CONCLUSIONS
Droplet digital polymerase chain reaction analysis of uncommon pathogenic EGFR variants can yield unique and reproducible results. Recognition of atypical patterns in EGFR ddPCR testing can prompt confirmatory molecular testing and aid appropriate targeted therapy selection for patients with non-small cell lung cancer.
Topics: Humans; Lung Neoplasms; ErbB Receptors; Carcinoma, Non-Small-Cell Lung; Polymerase Chain Reaction; Mutation; Female; Male; Middle Aged; Aged; High-Throughput Nucleotide Sequencing; Exons; Adult; DNA Mutational Analysis; Aged, 80 and over
PubMed: 37639432
DOI: 10.5858/arpa.2023-0088-OA -
Food Research International (Ottawa,... Dec 2023Viruses are major pathogens that cause food poisoning when ingested via contaminated food and water. Therefore, the development of foodborne virus detection technologies... (Review)
Review
Viruses are major pathogens that cause food poisoning when ingested via contaminated food and water. Therefore, the development of foodborne virus detection technologies that can be applied throughout the food distribution chain is essential for food safety. A common nucleic acid-based detection method is polymerase chain reaction (PCR), which has become the gold standard for monitoring food contamination by viruses due to its high sensitivity, and availability of commercial kits. However, PCR-based methods are labor intensive and time consuming, and are vulnerable to inhibitors that may be present in food samples. In addition, the methods are restricted with regard to site of analysis due to the requirement of expensive and large equipment for sophisticated temperature regulation and signal analysis procedures. To overcome these limitations, optical and electrical readout biosensors based on nucleic acid isothermal amplification technology and nanomaterials have emerged as alternatives for nucleic acid-based detection of foodborne viruses. Biosensors are promising portable detection tools owing to their easy integration into compact platforms and ability to be operated on-site. However, the complexity of food components necessitates the inclusion of tedious preprocessing steps, and the lack of stability studies on residual food components further restricts the practical application of biosensors as a universal detection method. Here, we summarize the latest advances in nucleic acid-based strategies for the detection of foodborne viruses, including PCR-based and isothermal amplification-based methods, gene amplification-free methods, as well as food pretreatment methods. The principles, strengths/disadvantages, and performance of each method, problems to be solved, and future prospects for the development of a universal detection method are discussed.
Topics: Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Food Safety; Viruses; Nucleic Acids
PubMed: 37986417
DOI: 10.1016/j.foodres.2023.113502 -
ACS Sensors Jul 2023Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of...
Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).
Topics: Humans; In Situ Hybridization, Fluorescence; Viruses; Polymerase Chain Reaction; Virus Diseases; Virion
PubMed: 37368999
DOI: 10.1021/acssensors.3c00239