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Biosensors & Bioelectronics Feb 2024The limit of detection (LOD), speed, and cost of crucial COVID-19 diagnostic tools, including lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and... (Review)
Review
The limit of detection (LOD), speed, and cost of crucial COVID-19 diagnostic tools, including lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reactions (PCR), have all improved because of the financial and governmental support for the epidemic. The most notable improvement in overall efficiency among them has been seen with PCR. Its significance for human health increased during the COVID-19 pandemic, when it emerged as the commonly used approach for identifying the virus. However, because of problems with speed, complexity, and expense, PCR deployment in point-of-care settings continues to be difficult. Microfluidic platforms offer a promising solution by enabling the development of smaller, more affordable, and faster PCR systems. In this review, we delve into the engineering challenges associated with the advancement of high-speed microfluidic PCR equipment. We introduce criteria that facilitate the evaluation and comparison of factors such as speed, LOD, cycling efficiency, and multiplexing capacity, considering sample volume, fluidics, PCR reactor geometry and materials, as well as heating/cooling methods. We also provide a comprehensive list of commercially available PCR devices and conclude with projections and a discussion regarding the current obstacles that need to be addressed in order to progress further in this field.
Topics: Humans; COVID-19; Pandemics; Biosensing Techniques; Polymerase Chain Reaction; Microfluidics; COVID-19 Testing
PubMed: 38039729
DOI: 10.1016/j.bios.2023.115830 -
Biosensors Apr 2024RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation.... (Review)
Review
RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation. Therefore, RNA detection technology has been widely used in many fields, especially in disease diagnosis, medical research, genetic engineering and other fields. However, the current RT-qPCR for RNA detection is complex, costly and requires the support of professional technicians, resulting in it not having great potential for rapid application in the field. PCR-free techniques are the most attractive alternative. They are a low-cost, simple operation method and do not require the support of large instruments, providing a new concept for the development of new RNA detection methods. This article reviews current PCR-free methods, overviews reported RNA biosensors based on electrochemistry, SPR, microfluidics, nanomaterials and CRISPR, and discusses their challenges and future research prospects in RNA detection.
Topics: Biosensing Techniques; RNA; Humans; Electrochemical Techniques; Polymerase Chain Reaction; Nanostructures; Surface Plasmon Resonance; Microfluidics
PubMed: 38667193
DOI: 10.3390/bios14040200 -
Clinical Infectious Diseases : An... Jul 2023A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive polymerase chain reaction on a rectal swab...
A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive polymerase chain reaction on a rectal swab for Monkeypox virus after a recent trip to Brazil, where he engaged in condomless sexual intercourse with multiple male partners.
Topics: Humans; Male; Mpox (monkeypox); Reinfection; Brazil; Monkeypox virus; Polymerase Chain Reaction
PubMed: 36905148
DOI: 10.1093/cid/ciad147 -
Virology Journal Nov 2023Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic...
BACKGROUND
Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations.
METHODS
The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1-H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV).
RESULTS
The results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/μL and 12 copies/μL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field.
CONCLUSION
An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.
Topics: Animals; Influenza in Birds; CRISPR-Cas Systems; Influenza A virus; Birds; Poultry; Sensitivity and Specificity; Real-Time Polymerase Chain Reaction; Newcastle disease virus; RNA
PubMed: 37957729
DOI: 10.1186/s12985-023-02232-7 -
Journal of Clinical Microbiology Oct 2023Rapid identification of the causative pathogens of central nervous system infections is essential for providing appropriate management and improving patient outcomes....
Rapid identification of the causative pathogens of central nervous system infections is essential for providing appropriate management and improving patient outcomes. The performance of QIAstat-Dx Meningitis/Encephalitis (ME) Panel-a multiplex PCR testing platform-in detecting pathogens implicated in meningitis and/or encephalitis was evaluated using BioFire FilmArray ME Panel as a comparator method. This multicenter study analyzed 585 retrospective residual cerebrospinal fluid specimens and 367 contrived specimens. The QIAstat-Dx ME Panel showed positive percent agreement (PPA) values of 100% for , , K1, , and / on clinical samples compared to the BioFire FilmArray ME Panel. The PPA values observed for and were 80% and 88.24%, respectively. Negative percent agreement (NPA) values were >99.0% for each of the six bacterial targets and one fungal target tested with clinical samples. One viral target, herpes simplex virus 1, exhibited a PPA value of 100.0%, while the remaining viral targets-human parechovirus, herpes simplex virus 2, human herpes virus 6, and varicella zoster virus-were >90.0%, with the exception of enterovirus, which had a PPA value of 77.8%. The QIAstat-Dx ME Panel detected five true-positive and four true-negative cases compared to BioFire FilmArray ME Panel. The NPA values for all viral pathogens were >99.0%. Overall, the QIAstat-Dx ME Panel showed comparable performance to the BioFire FilmArray ME Panel as a rapid diagnostic tool for community-acquired meningitis and encephalitis.
Topics: Humans; Multiplex Polymerase Chain Reaction; Retrospective Studies; Meningitis; Encephalitis; Meningoencephalitis
PubMed: 37702495
DOI: 10.1128/jcm.00426-23 -
Scientific Reports May 2024Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of...
Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.
Topics: SARS-CoV-2; Software; Polymerase Chain Reaction; Oligonucleotides; COVID-19; Computational Biology; DNA, Single-Stranded
PubMed: 38750104
DOI: 10.1038/s41598-024-59497-3 -
The Journal of Maternal-fetal &... Dec 2023The aim of this study was to evaluate if screening Group B colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic...
OBJECTIVES
The aim of this study was to evaluate if screening Group B colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic prophylaxis, compared with antepartum culture screening and analyze the sensitivity and specificity of polymerase chain reaction test.
METHODS
198 pregnant women with Group B colonization antepartum culture screening were included. When they arrived at hospital for delivery, two rectovaginal swabs were collected: for culture and polymerase chain reaction method.
RESULTS
The rate of Group B colonization antepartum detected by culture was 16.7%; at delivery was 17.2% when detected by culture and 19.7% using polymerase chain reaction method. The rate of inconclusive polymerase chain reaction tests was 0.5%. Considering intrapartum culture screening as gold standard, sensitivity and specificity of polymerase chain reaction test for intrapartum Group B colonization was 97.1% and 95.7%, respectively. The global rate of discordance between antepartum and intrapartum Group B colonization was 6.6%. The rate of women not treated with intrapartum antibiotic prophylaxis in the setting of positive intrapartum culture was significantly lower using intrapartum polymerase chain reaction test (0.5%) than with antepartum culture method (3.5%, = 0.035).
CONCLUSION
The use of intrapartum antibiotic prophylaxis can be more efficient when screening Group B colonization intrapartum by polymerase chain reaction test. Polymerase chain reaction method had a good performance in our study, with high sensitivity and specificity.
Topics: Pregnancy; Female; Humans; Pregnancy Complications, Infectious; Streptococcal Infections; Polymerase Chain Reaction; Parturition; Streptococcus agalactiae; Vagina
PubMed: 37766418
DOI: 10.1080/14767058.2023.2262078 -
International Journal of Public Health 2023We aimed to assess the association between rapid antigen detection tests and real-time reverse transcription-polymerase chain reaction assay for severe acute... (Meta-Analysis)
Meta-Analysis Review
Association Between Rapid Antigen Detection Tests and Real-Time Reverse Transcription-Polymerase Chain Reaction Assay for SARS-CoV-2: A Systematic Review and Meta-Analyses.
We aimed to assess the association between rapid antigen detection tests and real-time reverse transcription-polymerase chain reaction assay for severe acute respiratory syndrome coronavirus 2. We searched PubMed, Cochrane Library, EMBASE, and the Web of Science from their inception to 31 May 2023. A random-effects meta-analysis was used to estimate false positives in the RADTs group, relative to those in the RT-PCR group, and subgroup analyses were conducted based on the different Ct value cut-offs (<40 or ≥40). We performed this study in accordance with the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Fifty-one studies were included and considered to be of moderate quality. We found a satisfactory overall false positive rate (0.01, 95% CI: 0.00-0.01) for the RADTs compared to RT-PCR. In the stratified analysis, we also found that the false positive rates of the RADTs did not increase when Ct values of RT-PCR (Ct < 40, 0.01, 95% CI: 0.00-0.01; Ct ≥ 40, 0.01, 95% CI: 0.00-0.01). In conclusion, the best available evidence supports an association between RADTs and RT-PCR. When Ct-values were analyzed using cut-off <40 or ≥40, this resulted in an estimated false positive rate of only 1%.
Topics: Humans; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; SARS-CoV-2; COVID-19; COVID-19 Testing
PubMed: 37588042
DOI: 10.3389/ijph.2023.1605452 -
Genes Dec 2023The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV...
The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at -80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 10 copies/μL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and -20 °C and for 12 months at a temperature of -80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.
Topics: Humans; Rhinovirus; Polymerase Chain Reaction; Respiratory System; Nucleic Acid Amplification Techniques; RNA, Viral
PubMed: 38137032
DOI: 10.3390/genes14122210 -
Scientific Reports Nov 2023Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3...
Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50-60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.
Topics: Animals; Humans; RNA; Polymerase Chain Reaction; RNA, Messenger; Cell Line; Biological Assay; Real-Time Polymerase Chain Reaction; Mammals
PubMed: 37923834
DOI: 10.1038/s41598-023-46314-6