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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 -
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 -
Nature Methods May 2024
Topics: DNA; Humans; Multiplex Polymerase Chain Reaction
PubMed: 38745075
DOI: 10.1038/s41592-024-02287-6 -
Food Chemistry Aug 2024Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to...
Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCR-based method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 × 10 ng/μL vs. 1.36 × 10) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety.
Topics: Amanita; DNA, Fungal; Real-Time Polymerase Chain Reaction; DNA Primers; Polymerase Chain Reaction; China; Mushroom Poisoning
PubMed: 38520990
DOI: 10.1016/j.foodchem.2024.139086 -
Biochemistry and Molecular Biology... 2023The polymerase chain reaction (PCR) technique is one of the most potent tools in molecular biology. It is extensively used for various applications ranging from medical... (Review)
Review
The polymerase chain reaction (PCR) technique is one of the most potent tools in molecular biology. It is extensively used for various applications ranging from medical diagnostics to forensic science and food quality testing. This technique has facilitated to survive COVID-19 pandemic by identifying the virus-infected individuals effortlessly and effectively. This review explores the principles, recent advancements, challenges, and alternatives of PCR technique in the context of COVID-19 and fungal infections. The introduction of PCR technique for anyone new to this field is the primary aim of this review and thereby equips them to understand the science of COVID-19 and related fungal infections in a simplistic manner.
Topics: Humans; COVID-19; Pandemics; Polymerase Chain Reaction; SARS-CoV-2; Mycoses; COVID-19 Testing
PubMed: 37485773
DOI: 10.1002/bmb.21771 -
Journal of the American Academy of... Oct 2023
Topics: Humans; Prospective Studies; Mpox (monkeypox); Skin; Polymerase Chain Reaction; Oropharynx
PubMed: 37295504
DOI: 10.1016/j.jaad.2023.05.071 -
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 -
Chinese Medical Journal Mar 2024Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be...
BACKGROUND
Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.
METHODS
The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed.
RESULTS
The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts.
CONCLUSIONS
This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.
Topics: Humans; HIV-1; DNA, Viral; Reproducibility of Results; Polymerase Chain Reaction; HIV Infections; Real-Time Polymerase Chain Reaction
PubMed: 38433332
DOI: 10.1097/CM9.0000000000003081 -
Methods in Molecular Biology (Clifton,... 2024DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of...
DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of learning, and DNA barcoding method allows to merge necessary-to-study experimental techniques such as DNA isolation and purification, PCR, and basic BLAST search into a two- or three-week inquiry-based student project. It also provides a research-based experience to the students, who, when organized in groups, can design their own DNA-barcoding project if they wish. Here, we describe how DNA barcoding can be offered in an undergraduate college or advanced high school settings. This chapter is intended to help college and high school instructors to include DNA barcoding in their classes.
Topics: DNA Barcoding, Taxonomic; Students; Universities; DNA; Humans; Curriculum; Polymerase Chain Reaction
PubMed: 38683341
DOI: 10.1007/978-1-0716-3581-0_33 -
Poultry Science Aug 2023Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant...
Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 × 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent specificity, sensitivity, and throughput.
Topics: Animals; Poultry; Reverse Transcriptase Polymerase Chain Reaction; Reproducibility of Results; Reverse Transcription; Chickens; Sensitivity and Specificity; Bird Diseases; Virus Diseases; Respiratory Tract Infections; Polymerase Chain Reaction; Multiplex Polymerase Chain Reaction; Poultry Diseases
PubMed: 37354617
DOI: 10.1016/j.psj.2023.102852