-
Journal of Bioscience and Bioengineering Jul 2022A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive...
A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.
Topics: DNA Primers; Haptens; Immunologic Tests; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 35450786
DOI: 10.1016/j.jbiosc.2022.03.006 -
Biosensors & Bioelectronics Sep 2019Over the last decade, nucleic acid amplification tests (NAATs) including polymerase chain reaction (PCR) were an indispensable methodology for diagnosing cancers, viral... (Review)
Review
Over the last decade, nucleic acid amplification tests (NAATs) including polymerase chain reaction (PCR) were an indispensable methodology for diagnosing cancers, viral and bacterial infections owing to their high sensitivity and specificity. Because the NAATs can recognize and discriminate even a few copies of nucleic acid (NA) and species-specific NA sequences, NAATs have become the gold standard in a wide range of applications. However, limitations of NAAT approaches have recently become more apparent by reason of their lengthy run time, large reaction volume, and complex protocol. To meet the current demands of clinicians and biomedical researchers, new NAATs have developed to achieve ultrafast sample-to-answer protocols for the point-of-care testing (POCT). In this review, ultrafast NA-POCT platforms are discussed, outlining their NA amplification principles as well as delineating recent advances in ultrafast NAAT applications. The main focus is to provide an overview of NA-POCT platforms in regard to sample preparation of NA, NA amplification, NA detection process, interpretation of the analysis, and evaluation of the platform design. Increasing importance will be given to innovative, ultrafast amplification methods and tools which incorporate artificial intelligence (AI)-associated data analysis processes and mobile-healthcare networks. The future prospects of NA POCT platforms are promising as they allow absolute quantitation of NA in individuals which is essential to precision medicine.
Topics: Animals; Artificial Intelligence; Biosensing Techniques; Equipment Design; Humans; Nucleic Acid Amplification Techniques; Nucleic Acids; Point-of-Care Systems; Polymerase Chain Reaction; Time Factors
PubMed: 31252258
DOI: 10.1016/j.bios.2019.111448 -
The Journal of Applied Laboratory... Jan 2024Digital polymerase chain reaction (dPCR) is an accurate and sensitive molecular method that can be used in clinical diagnostic, prognostic, and predictive tests. The key...
BACKGROUND
Digital polymerase chain reaction (dPCR) is an accurate and sensitive molecular method that can be used in clinical diagnostic, prognostic, and predictive tests. The key component of the dPCR method is the partitioning of a single reaction into many thousands of droplets, nanochannels or other nano- or picoliter-sized reactions. This results in high enough sensitivity to detect rare nucleic acid targets and provides an absolute quantification of target sequences or alleles compared to other PCR-based methods.
CONTENT
An increasing number of dPCR platforms have been introduced commercially in recent years and more are being developed. These platforms differ in the method of partitioning, degree of automation, and multiplexing capabilities but all can be used in similar ways for sensitive and highly accurate quantification of a variety of nucleic acid targets. Currently, clinical applications of dPCR include oncology, microbiology and infectious disease, genetics, and prenatal/newborn screening. Commercially available tests for clinical applications are being developed for variants with diagnostic, prognostic, and therapeutic significance in specific disease types.
SUMMARY
The power of dPCR technology relies on the partitioning of the reactions and results in increased sensitivity and accuracy compared to qPCR. More recently, the sensitivity of dPCR has been applied to the detection of known variants in cell-free DNA and circulating tumor DNA. Future clinical applications of dPCR include liquid biopsy, treatment resistance detection, screening for minimal residual disease, and monitoring allograft engraftment in transplanted patients.
Topics: Pregnancy; Female; Infant, Newborn; Humans; Polymerase Chain Reaction; Prenatal Diagnosis; Nucleic Acids
PubMed: 38167753
DOI: 10.1093/jalm/jfad103 -
Trends in Biochemical Sciences Feb 2020
Review
Topics: Photons; Polymerase Chain Reaction; Temperature
PubMed: 31866304
DOI: 10.1016/j.tibs.2019.11.007 -
Molekuliarnaia Biologiia 2023This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the... (Review)
Review
This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the enrichment of target genomic fragments. The methods of molecular hybridization, polymerase chain reaction, and CRISPR-Cas9 system for this purpose are considered. Examples of the practical use of the developed methods for research and diagnostic purposes are given.
Topics: CRISPR-Cas Systems; DNA; Polymerase Chain Reaction; Genome; High-Throughput Nucleotide Sequencing
PubMed: 37326047
DOI: No ID Found -
BioTechniques Jul 2019
Topics: Animals; Equipment Design; Humans; Miniaturization; Polymerase Chain Reaction; Time Factors
PubMed: 31238703
DOI: 10.2144/btn-2019-0076 -
Cold Spring Harbor Protocols Mar 2022The polymerase chain reaction (PCR) can be used to produce both nonradiolabeled DNA probes and radiolabeled DNA probes with high specific activity. In this protocol, PCR...
The polymerase chain reaction (PCR) can be used to produce both nonradiolabeled DNA probes and radiolabeled DNA probes with high specific activity. In this protocol, PCR is used to generate double-stranded probes. Related methods, including the generation of asymmetric probes by PCR, are also discussed.
Topics: DNA; DNA Probes; Polymerase Chain Reaction
PubMed: 34907078
DOI: 10.1101/pdb.prot100610 -
Japanese Journal of Ophthalmology Jan 2017Uveitis is a sight-threatening intraocular inflammatory disorder which may occur from both infectious and non-infectious or autoimmune causes. The frequency of... (Review)
Review
Uveitis is a sight-threatening intraocular inflammatory disorder which may occur from both infectious and non-infectious or autoimmune causes. The frequency of infectious uveitis and autoimmune uveitis varies depending on countries and regions. According to a nationwide survey conducted by the Japanese Ocular Inflammation Society, infectious and non-infectious uveitis accounted for 16.4 and 50.1% of new patients, respectively while the remaining 33.5% of new uveitis cases were not classified or were idiopathic uveitis. Infectious uveitis is particularly important because it causes tissue damage to the eye and may result in blindness unless treated. However, it can be treated if the pathogenic microorganisms are identified promptly and accurately. Remarkable advancements in molecular and immunological technologies have been made in the last decade, and the diagnosis of infectious uveitis has been greatly improved by the application of molecular and immunological investigations, particularly polymerase chain reaction (PCR). PCR performed on a small amount of ocular samples provides a prompt, sensitive, and specific molecular diagnosis of pathogenic microorganisms in the eye. This technology has opened a new era in the diagnosis and treatment of uveitis, enabling physicians to establish new clinical entities of uveitis caused by infectious microorganisms, identify pathogens in the eyes of many patients with uveitis, and determine prompt diagnosis and appropriate therapy. Here we review the PCR process, new PCR tests specialized for ocular diseases, microorganisms detected by the PCR tests, diseases in the eye caused by these microorganisms, and the clinical characteristics, diagnosis, and therapy of uveitis.
Topics: DNA; Humans; Polymerase Chain Reaction; Uveitis
PubMed: 27787641
DOI: 10.1007/s10384-016-0474-9 -
BMC Ophthalmology Oct 2016To study the value and safety of aqueous humor polymerase chain reaction (PCR) analysis for Herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr virus and...
BACKGROUND
To study the value and safety of aqueous humor polymerase chain reaction (PCR) analysis for Herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr virus and Toxoplasma gondii in patients with uveitis.
METHODS
Records of 45 consecutive patients with anterior and posterior uveitis who underwent AC paracentesis with PCR were reviewed. The main outcome measure was frequency of PCR positivity. Secondary outcomes were alteration of treatment, safety of paracentesis, and correlation of keratitic precipitates with PCR positivity, RESULTS: The overall PCR positivity was 48.9 % (22/45). Therapy was changed because of the PCR results in 14/45 patients (37.7 %). One patient experienced a paracentesis related complication (1/45, 2.2 %) without long-term sequelae.
CONCLUSION
Aqueous PCR altered the diagnosis and treatment in over a third of our patients and was relatively safe. Aqueous PCR should be considered for uveitis of atypical clinical appearance, recurrent severe uveitis of uncertain etiology, and therapy refractory cases.
Topics: Adult; Aged; Aqueous Humor; Diagnostic Techniques, Ophthalmological; Eye Infections, Viral; Female; Humans; Male; Middle Aged; Polymerase Chain Reaction; Toxoplasmosis; Uveitis; Young Adult
PubMed: 27793120
DOI: 10.1186/s12886-016-0369-z -
Cold Spring Harbor Protocols Jun 2019Polymerase chain reaction (PCR), rather than the more labor-intensive minipreps, is often used to screen colonies of for recombinant plasmids containing sequences of...
Polymerase chain reaction (PCR), rather than the more labor-intensive minipreps, is often used to screen colonies of for recombinant plasmids containing sequences of interest. When screening colonies for a DNA fragment amplified by PCR, the same primers used in the amplification reaction can be used for screening. To discover the orientation of the cloned fragment, a third primer can be used that binds to the plasmid sequences immediately adjacent to the insertion site and points toward the insert.
Topics: Bacteria; Colony Count, Microbial; Polymerase Chain Reaction
PubMed: 31160384
DOI: 10.1101/pdb.prot095224