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Vox Sanguinis Dec 2023In Japan, the prevalence of haptoglobin deficiency is approximately 1 in 4000. Haptoglobin-deficient individuals may produce anti-haptoglobin from allo-immunization,...
BACKGROUND AND OBJECTIVES
In Japan, the prevalence of haptoglobin deficiency is approximately 1 in 4000. Haptoglobin-deficient individuals may produce anti-haptoglobin from allo-immunization, leading to serious transfusion reactions. Therefore, implementation of a consistent supply of haptoglobin-deficient fresh frozen plasma is crucial. We developed a novel reagent to facilitate large-scale identification of haptoglobin-deficient individuals as potential donors of plasma products.
MATERIALS AND METHODS
We established mouse monoclonal anti-haptoglobin-producing cell lines (three clones) using the hybridoma method by immunizing mice with the haptoglobin protein. Purified antibodies were conjugated with carboxylate-modified polystyrene latex beads and used for haptoglobin measurements by the latex agglutination method using an automatic analyser (LABOSPECT008). Samples with low protein concentrations were re-examined by enzyme-linked immunosorbent assay to confirm the results. Additionally, the haptoglobin gene was amplified by polymerase chain reaction to confirm the haptoglobin deletion allele (Hp ).
RESULTS
From February to October 2022, 7476 blood donor samples were screened. Two haptoglobin-deficient and 21 low-haptoglobin-expressing individuals were identified. Two haptoglobin-deficient donors were found homozygous for Hp , and 19 (90%) of the 21 low-haptoglobin-expressing individuals were heterozygous for Hp , which includes the first reported case of heterozygous Hp /Hp .
CONCLUSION
We developed a new reagent for the detection of haptoglobin deficiency, which is automatable and inexpensive and appears useful for large-scale screening of blood donors.
Topics: Animals; Humans; Mice; Blood Donors; Enzyme-Linked Immunosorbent Assay; Haptoglobins; Heterozygote; Polymerase Chain Reaction; Antibodies, Monoclonal
PubMed: 37798623
DOI: 10.1111/vox.13543 -
Diagnostic Microbiology and Infectious... Oct 2023Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and...
Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and diagnostic delays can lead to irreversible damage to the vision. The gold standard for diagnosis has been a sample culture that lasts approximately 2 weeks. Nevertheless, the essence of time has led to the need for an accurate and fast technique to detect A. castellanii from a sample. We developed both traditional and quantitative real-time-PCR-based methods to detect A. castellanii in less than 3 hours and with the sensitivity of one amoeba. Diagnostic laboratories can select the best-suited method for their purposes from 2 comparable methods. The correct treatment can be initiated from the emergency room when the diagnosis has been made quickly within a few hours, hence saving the patient from long-term complications.
Topics: Humans; Acanthamoeba castellanii; Rapid Diagnostic Tests; Acanthamoeba Keratitis; Cornea; Real-Time Polymerase Chain Reaction
PubMed: 37506594
DOI: 10.1016/j.diagmicrobio.2023.116014 -
BMC Biotechnology Apr 2024Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase...
Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10 bp and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.
Topics: Polymerase Chain Reaction; DNA-Directed DNA Polymerase; Uracil; Plasmids; DNA
PubMed: 38566117
DOI: 10.1186/s12896-024-00844-7 -
Mikrochimica Acta Dec 2023The current use of the single serum biomarker α-fetoprotein (AFP) in clinical practice has limitations in terms of specificity and sensitivity. We propose a strategy...
The current use of the single serum biomarker α-fetoprotein (AFP) in clinical practice has limitations in terms of specificity and sensitivity. We propose a strategy that combines antigen capture polymerase chain reaction (AC-PCR), lateral flow assay (LFA), and electrochemical biosensors to detect both AFP and circulating tumor cells (CTCs) in liver cancer serum. First, we used the AC-PCR technique to achieve target separation, purification, signal conversion, and amplification, eliminating target heterogeneity. Then, we achieved rapid results through the LFA and electrochemical biosensor platforms. As a result, the proposed assay has limits of 5 cells/mL for CTCs and 5 µg/L for AFP. The proposed method was applied effectively to simulated blood samples. This method has the potential to play a role in early liver cancer and provide a potential application for the diagnosis and precision treatment of liver cancer.
Topics: Humans; Biomarkers, Tumor; alpha-Fetoproteins; Liver Neoplasms; Polymerase Chain Reaction; Biosensing Techniques
PubMed: 38091092
DOI: 10.1007/s00604-023-06098-y -
British Medical Bulletin Sep 2023Fungal disease has historically presented a diagnostic challenge due to its often non-specific clinical presentations, relative infrequency and reliance on insensitive...
INTRODUCTION
Fungal disease has historically presented a diagnostic challenge due to its often non-specific clinical presentations, relative infrequency and reliance on insensitive and time-intensive fungal culture.
SOURCES OF DATA
We present the recent developments in fungal diagnostics in the fields of serological and molecular diagnosis for the most clinically relevant pathogens; developments that have the potential to revolutionize fungal diagnosis through improvements in speed, simplicity and sensitivity. We have drawn on a body of evidence including recent studies and reviews demonstrating the effectiveness of antigen and antibody detection and polymerase chain reaction (PCR) in patients with and without concurrent human immunodeficiency virus infection.
AREAS OF AGREEMENT
This includes recently developed fungal lateral flow assays, which have a low cost and operator skill requirement that give them great applicability to low-resource settings. Antigen detection for Cryptococcus, Histoplasma and Aspergillus spp. are much more sensitive than culture. PCR for Candida spp., Aspergillus spp., Mucorales and Pneumocystis jirovecii is more sensitive than culture and usually faster.
AREAS OF CONTROVERSY
Effort must be made to utilize recent developments in fungal diagnostics in clinical settings outside of specialist centres and integrate their use into standard medical practice. Given the clinical similarities of the conditions and frequent co-infection, further study is required into the use of serological and molecular fungal tests, particularly in patients being treated for tuberculosis.
GROWING POINTS
Further study is needed to clarify the utility of these tests in low-resource settings confounded by a high prevalence of tuberculosis.
AREAS TIMELY FOR DEVELOPING RESEARCH
The diagnostic utility of these tests may require revision of laboratory work flows, care pathways and clinical and lab coordination, especially for any facility caring for the immunosuppressed, critically ill or those with chronic chest conditions, in whom fungal disease is common and underappreciated.
Topics: Humans; Mycoses; Candida; Polymerase Chain Reaction
PubMed: 37328942
DOI: 10.1093/bmb/ldad011 -
Archives of Pathology & Laboratory... Mar 2024Thalassemia is the most widely distributed monogenic autosomal recessive disorder in the world. Accurate genetic analysis of thalassemia is crucial for thalassemia...
CONTEXT.—
Thalassemia is the most widely distributed monogenic autosomal recessive disorder in the world. Accurate genetic analysis of thalassemia is crucial for thalassemia prevention.
OBJECTIVE.—
To compare the clinical utility of a third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles with routine polymerase chain reaction (PCR) in genetic analysis of thalassemia and explore the molecular spectrum of thalassemia in Hunan Province.
DESIGN.—
Subjects in Hunan Province were recruited, and hematologic testing was performed. Five hundred four subjects positive on hemoglobin testing were then used as the cohort, and third-generation sequencing and routine PCR were used for genetic analysis.
RESULTS.—
Of the 504 subjects, 462 (91.67%) had the same results, whereas 42 (8.33%) exhibited discordant results between the 2 methods. Sanger sequencing and PCR testing confirmed the results of third-generation sequencing. In total, third-generation sequencing correctly detected 247 subjects with variants, whereas PCR identified 205, which showed an increase in detection of 20.49%. Moreover, α triplications were identified in 1.98% (10 of 504) hemoglobin testing-positive subjects in Hunan Province. Seven hemoglobin variants with potential pathogenicity were detected in 9 hemoglobin testing-positive subjects.
CONCLUSIONS.—
Third-generation sequencing is a more comprehensive, reliable, and efficient approach for genetic analysis of thalassemia than PCR, and allowed for a characterization of the thalassemia spectrum in Hunan Province.
Topics: Humans; Thalassemia; Hematologic Tests; Blood Coagulation Tests; Polymerase Chain Reaction; Hemoglobins; Mutation; Genotype; beta-Thalassemia
PubMed: 37270807
DOI: 10.5858/arpa.2022-0299-OA -
Analytical Chemistry Dec 2023This paper presents the design, microfabrication, and demonstration of a novel microfluidic grinding mill for the lysis of the dinoflagellate, , a neurotoxin-producing...
This paper presents the design, microfabrication, and demonstration of a novel microfluidic grinding mill for the lysis of the dinoflagellate, , a neurotoxin-producing genus of algae that is responsible for red tide and paralytic shellfish poisoning. The mill consists of a high-speed, hydrodynamically driven microrotor coupled to a micro grinding mill that lyses robust algal cells by mechanical abrasion with single-pass efficiencies as high as 97%. These efficiencies are comparable to, or better than, current mechanical and chemical lysing methods without adding complications associated with harsh chemical additives that can interfere with subsequent downstream bioanalysis. Release of cytoplasm from lysed algae was confirmed using polymerase chain reaction (PCR) amplification of DNA using dinoflagellate primers.
Topics: Dinoflagellida; Polymerase Chain Reaction; DNA Primers
PubMed: 37976075
DOI: 10.1021/acs.analchem.3c02344 -
Nature Biomedical Engineering Dec 2023The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the...
The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.
Topics: Humans; Mutation; Neoplasms; Polymerase Chain Reaction; DNA; Nucleotides
PubMed: 37500748
DOI: 10.1038/s41551-023-01072-8 -
Analytical Chemistry Dec 2023Microfluidics can split samples into thousands or millions of partitions, such as droplets or nanowells. Partitions capture analytes according to a Poisson distribution,...
Microfluidics can split samples into thousands or millions of partitions, such as droplets or nanowells. Partitions capture analytes according to a Poisson distribution, and in diagnostics, the analyte concentration is commonly inferred with a closed-form solution via maximum likelihood estimation (MLE). Here, we present a new scalable approach to multiplexing analytes. We generalize MLE with microfluidic partitioning and extend our previously developed Sparse Poisson Recovery (SPoRe) inference algorithm. We also present the first in vitro demonstration of SPoRe with droplet digital PCR (ddPCR) toward infection diagnostics. Digital PCR is intrinsically highly sensitive, and SPoRe helps expand its multiplexing capacity by circumventing its channel limitations. We broadly amplify bacteria with 16S ddPCR and assign barcodes to nine pathogen genera by using five nonspecific probes. Given our two-channel ddPCR system, we measured two probes at a time in multiple groups of droplets. Although individual droplets are ambiguous in their bacterial contents, we recover the concentrations of bacteria in the sample from the pooled data. We achieve stable quantification down to approximately 200 total copies of the 16S gene per sample, enabling a suite of clinical applications given a robust upstream microbial DNA extraction procedure. We develop a new theory that generalizes the application of this framework to many realistic sensing modalities, and we prove scaling rules for system design to achieve further expanded multiplexing. The core principles demonstrated here could impact many biosensing applications with microfluidic partitioning.
Topics: Microfluidics; Polymerase Chain Reaction; Bacteria
PubMed: 37971927
DOI: 10.1021/acs.analchem.3c01176 -
Laboratory Medicine Mar 2024Different mitochondrial DNA genotypes can coexist in a cell population as well as in a single cell, a condition known as heteroplasmy. Here, we accurately determined the...
OBJECTIVE
Different mitochondrial DNA genotypes can coexist in a cell population as well as in a single cell, a condition known as heteroplasmy. Here, we accurately determined the heteroplasmy levels of the m.3243A>G mutation, which is the most frequently identified mutation in patients with mitochondrial diseases, using droplet digital polymerase chain reaction (ddPCR).
METHODS
The m.3243A>G heteroplasmy levels in artificial heteroplasmy controls mixed with various proportions of wild-type and mutant plasmids were measured using ddPCR, PCR-restriction fragment length polymorphism, and Sanger sequencing. The m.3243A>G heteroplasmy levels in DNA, extracted from the peripheral blood of patients with suspected mitochondrial disease and healthy subjects, were determined using ddPCR.
RESULTS
The accuracy of the ddPCR method was high. The lower limit of detection was 0.1%, which indicated its higher sensitivity compared with other methods. The m.3243A>G heteroplasmy levels in peripheral blood, measured using ddPCR, correlated inversely with age at the time of analysis. The m.3243A>G mutation may be overlooked in the peripheral blood-derived DNA of elderly people, as patients >60 years of age have heteroplasmy levels <10%, which is difficult to detect using methods other than the highly sensitive ddPCR.
CONCLUSION
ddPCR may be considered an accurate and sensitive method for measuring m.3243 A>G heteroplasmy levels of mitochondrial DNA.
Topics: Humans; Aged; Mutation; DNA, Mitochondrial; Mitochondrial Diseases; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length
PubMed: 37478467
DOI: 10.1093/labmed/lmad063