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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 -
European Review For Medical and... Nov 2023The study aimed to evaluate respiratory virus infections in adult patients with hematological malignancies (HM).
OBJECTIVE
The study aimed to evaluate respiratory virus infections in adult patients with hematological malignancies (HM).
PATIENTS AND METHODS
The medical records of patients who were followed up by the hematology clinic at Başakşehir Çam and Sakura City Hospital between March 2021 and March 2023 with a diagnosis of HM and who underwent real-time polymerase chain reaction (RT-PCR) testing for nasopharyngeal/oropharyngeal specimens taken with suspected respiratory tract infection constituted the study data.
RESULTS
Infections were symptomatic in 64.56% of patients, and the most common symptoms were fever (48.10%) and cough (18.99%). The mortality rate was 25.32% over a two-year period. When the samples were examined, positive test frequency was 43.04%, and the three most common pathogens were Influenza A (10.13%), SARS-CoV-2 (8.86%), and rhinovirus/enterovirus (7.59%). The frequency of positive tests from HMs was highest in patients with AML (p=0.042). Respiratory PCR kit positivity was higher in patients who had any symptoms (p=0.002) and cough (p=0.003). Test positivity was higher in patients with any pathological radiological finding (p=0.039) and ground glass appearance (p=0.010). The risk of death was found to be 5.848 times higher in patients with dyspnea compared to those without (OR: 5.848, 95% CI: 1.143-29.915, p=0.034).
CONCLUSIONS
Respiratory tract virus panel PCR test positivity is more common in patients with HM presenting with respiratory tract infection symptoms in the presence of AML diagnosis, symptomatic infection, cough, radiological findings, and ground glass appearance. Mortality risk is high in HM patients with respiratory tract virus infection who have shortness of breath.
Topics: Humans; Adult; Retrospective Studies; Cough; Viruses; Virus Diseases; Respiratory Tract Infections; Real-Time Polymerase Chain Reaction; Dyspnea; Hematologic Neoplasms; Leukemia, Myeloid, Acute
PubMed: 37975403
DOI: 10.26355/eurrev_202311_34358 -
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 -
Archivos Argentinos de Pediatria Oct 2023Introduction. The COVID-19 pandemic has brought to light the need for rapid diagnostic tests. The gold standard test is reverse transcription-polymerase chain reaction...
Introduction. The COVID-19 pandemic has brought to light the need for rapid diagnostic tests. The gold standard test is reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR requires equipment and trained personnel, and results may take a long waiting time. The BD Veritor® System is a rapid chromatographic method used for the detection of severe acute respiratory syndrome coronavirus 2 antigen in symptomatic individuals. The primary objective of this study is to assess the sensitivity and specificity of the antigen test (AT) compared to the RT-PCR in the pediatric population. Population and methods. Prospective study with a diagnostic test. All children younger than 17 years in the first 5 days of symptom onset, who consulted between July 2021 and February 2022, were included. A minimum of 300 specimens was estimated to achieve an accuracy of ±8.76% and ±3.68% for sensitivity and specificity, respectively. Specimens were analyzed in parallel using both methodologies. Results. Of 316 paired samples, 33 were positive by both methods; 6 were positive only by RT-PCR. The specificity of the AT was 100%; sensitivity was 84.6%, with a positive and negative predictive value of 100% and 98%, respectively. Conclusions. The AT proved to be useful in the diagnosis of pediatric patients with COVID-19 in the first 5 days of symptom onset, although those with a negative AT and high clinical suspicion should confirm their result with a RT-PCR. Clinical trial registration: PRIISA.BA - Record number: 4912 - Date of registration: 07/07/2021.
Topics: Humans; Child; COVID-19; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Pandemics; Prospective Studies; Reverse Transcription; Sensitivity and Specificity; COVID-19 Testing
PubMed: 36883808
DOI: 10.5546/aap.2022-02908.eng -
Biomedical and Environmental Sciences :... Sep 2023To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.
OBJECTIVE
To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.
METHODS
A database of capsular polysaccharide ( ) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.
RESULTS
A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains.
CONCLUSION
A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.
Topics: Real-Time Polymerase Chain Reaction; Serotyping; Streptococcus pneumoniae; Serogroup
PubMed: 37803892
DOI: 10.3967/bes2023.078 -
Annali Di Igiene : Medicina Preventiva... 2024The introduction of the vaccine against SARS-CoV-2 has represented a cornerstone in the containment of the pandemic. Our aim was to assess the vaccination schedules in...
BACKGROUND
The introduction of the vaccine against SARS-CoV-2 has represented a cornerstone in the containment of the pandemic. Our aim was to assess the vaccination schedules in relation to the infection free interval and to the duration of positivity in case of infection.
STUDY DESIGN
This study involves the SARS-CoV-2 infected people managed by the Local Health Authority ASL 1 Abruzzo. The data collected included: vaccine administration date, vaccine type, information on the Polymerase Chain Reaction test positivity, and demographic variables, such as age and sex.
METHODS
The duration of Polymerase Chain Reaction test positivity was assessed in relation to the vaccination status, the vaccine type and the time interval between the last vaccination dose and the first nasopharyngeal positive swab over the considered period.
RESULTS
The infection duration (DAYS) was significantly shorter in subjects vaccinated with a booster dose than unvaccinated subjects (12.8 vs 14.6; p<0.0001) and subjects vaccinated with the primary series only (12.8 vs 14.1; p<0.0001). Duration of PCR positivity was shorter with heterologous immunisation than with other vaccination schedules (p=0.0317).
CONCLUSIONS
This study highlights, in a large cohort of patients, the association between vaccination schedule and the response to infection.
Topics: Humans; Immunization Schedule; COVID-19; COVID-19 Vaccines; SARS-CoV-2; Vaccines; Vaccination; Polymerase Chain Reaction; COVID-19 Testing
PubMed: 38386025
DOI: 10.7416/ai.2024.2613 -
The Veterinary Quarterly Dec 2023High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous...
High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 10 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.
Topics: Cats; Animals; Calicivirus, Feline; Caliciviridae Infections; Polymerase Chain Reaction; Vaccines; Mutation; Cat Diseases
PubMed: 37851857
DOI: 10.1080/01652176.2023.2272188 -
Molecular Genetics & Genomic Medicine Aug 2023Copy number variation sequencing (CNV-seq) could detect most chromosomal abnormalities except polyploidy, and quantitative fluorescence polymerase chain reaction...
BACKGROUND
Copy number variation sequencing (CNV-seq) could detect most chromosomal abnormalities except polyploidy, and quantitative fluorescence polymerase chain reaction (QF-PCR) is a supplementary method to CNV-seq in triploid detection. This study aimed to evaluate the feasibility of sequential application of CNV-seq and QF-PCR in genetic analysis of miscarriage and stillbirth.
METHODS
A total of 261 fetal specimens were analyzed by CNV-seq, and QF-PCR was only further performed for samples with normal female karyotype identified by CNV-seq. Cost and turnaround time (TAT) was analyzed for sequential detection strategy. Subgroup analysis and logistic regression were carried out to evaluate the relationship between clinical characteristics (maternal age, gestational age, and number of pregnancy losses) and the occurrence of chromosomal abnormalities.
RESULTS
Abnormal results were obtained in 120 of 261 (45.98%) cases. Aneuploidy was the most common abnormality (37.55%), followed by triploidy (4.98%) and pathogenic copy number variations (pCNVs) (3.45%). CNV-seq could detect the triploidy with male karyotype, and QF-PCR could further identify the remaining triploidy with female karyotype. In this study, we found more male triploidies than female triploidies. With the same ability in chromosomal abnormalities detection, the cost of sequential strategy decreased by 17.35% compared with combined strategy. In subgroup analysis, significant difference was found in the frequency of total chromosomal abnormalities between early abortion group and late abortion group. Results of logistic regression showed a trend that pregnant women with advanced age, first-time abortion, and abortion earlier than 12 weeks were more likely to detect chromosomal aberrations in their products of conception.
CONCLUSION
Sequential application of CNV-seq and QF-PCR is an economic and practical strategy to identify chromosomal abnormalities in fetal tissue.
Topics: Female; Pregnancy; Male; Humans; Infant; Abortion, Spontaneous; DNA Copy Number Variations; Stillbirth; Triploidy; Chromosome Disorders; Chromosome Aberrations; Polymerase Chain Reaction
PubMed: 37073418
DOI: 10.1002/mgg3.2187 -
International Journal of Molecular... Sep 2023The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative...
The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase-DNA-dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease in PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity.
Topics: Humans; Polymerase Chain Reaction; DNA-Directed DNA Polymerase; Amino Acids; Dietary Fiber; Nucleotides
PubMed: 37686447
DOI: 10.3390/ijms241713643 -
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