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Archives of Razi Institute Jun 2023Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques...
Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques have been developed for its accurate diagnosis. However, they have some limitations. The present study was conducted to investigate the efficacy of specific modulation frequency (SMF), compared to other routine diagnostic methods for detecting . Blood samples were collected from 30 diseased cats suspected of having FIP based on clinical signs. Electrophoresis, polymerase chain reaction (PCR), and SMF tests were performed for each sample. The sensitivity and specificity of each test, as well as the agreement between the tests and the gold standard (the combination of PCR, electrophoresis, and bioresonance results), were calculated using the Kappa coefficient method. The sensitivity and specificity of electrophoresis, PCR, and SMF for the diagnosis of FIP were 70.6%, 70.6%, 100%, and 100%, 72.7%, 81.8%, respectively. According to the findings of the present study, SMF is effective and safe in FIP diagnosis, which is a challenge in veterinary medicine diagnosis.
Topics: Animals; Cats; Feline Infectious Peritonitis; Reverse Transcriptase Polymerase Chain Reaction; Polymerase Chain Reaction; Coronavirus, Feline; Electrophoresis; Cat Diseases
PubMed: 38028839
DOI: 10.22092/ARI.2023.360790.2606 -
BioTechniques Jan 2020
Topics: Polymerase Chain Reaction; Precision Medicine; Serial Publications
PubMed: 31939707
DOI: 10.2144/btn-2019-0162 -
Biochemistry and Molecular Biology... Jan 2022We present a course-embedded undergraduate research module that involves real-time polymerase chain reaction testing for the presence of SARS-CoV-2 in environmental...
An undergraduate laboratory experiment with real-world applications: Utilizing templateless polymerase chain reaction and real-time polymerase chain reaction to test for SARS-CoV-2 RNA.
We present a course-embedded undergraduate research module that involves real-time polymerase chain reaction testing for the presence of SARS-CoV-2 in environmental samples. A positive control RNA was constructed and two RNA extraction methods were compared and a range of primers were available to compare. Using a combination of published protocols, we assembled a successful project that illustrated a topical exercise similar to real-world assay development. The exercise is aimed at upper-level undergraduates and requires 3 weeks of laboratory periods. The students were able to design and test experimental protocols, while learning about RNA detection. This project could be utilized in upper-level classes including molecular biology, biochemistry, biotechnology, or for independent research projects.
Topics: COVID-19; Humans; Laboratories; RNA, Viral; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Students
PubMed: 34862844
DOI: 10.1002/bmb.21593 -
PloS One 2019Multigene-based PCR tests are time-consuming and limiting aspects of the protocol include increased risk of operator-based variation. In addition, such protocols are...
BACKGROUND
Multigene-based PCR tests are time-consuming and limiting aspects of the protocol include increased risk of operator-based variation. In addition, such protocols are complex to transfer and reproduce between laboratories.
AIMS
Evaluate the clinical utility of a pre-spotted PCR plate (PSP) for a novel multigene (n = 51) blood-based gene expression diagnostic assay for neuroendocrine tumors (NETs).
METHODS
A pilot study (n = 44; 8 controls and 36 NETs) was undertaken to compare CQ, normalized gene expression and algorithm-based output (NETest score). Gene expression was then evaluated between matched blood:tumor tissue samples (n = 7). Thereafter, two prospective sets (diagnostic: n = 167; clinical validation: n = 48, respectively) were evaluated for diagnostic and clinical utility value. Two independent molecular diagnostics facilities were used to assess assay reproducibility and inter-laboratory metrics. Samples were collected (per CLIA protocol) processed to mRNA and cDNA and then either run per standard assay (liquid primers) or on PSPs. Separately, matching plasma samples were analyzed for chromogranin A (CgA). Statistics included non-parametric testing, Pearson-concordance, Predictive Modeling and AUROC analyses.
RESULTS
In the pilot study (n = 44), CQ values were highly concordant (r: 0.82, p<0.0001) and normalized gene expression data significantly related (p<0.0001) (Pearson-pairwise correlation). NETest values were not different (49.7±33 standard vs. 48.5±31.5 PSP) and the overall concordance in output 96%. Predictive modelling confirmed this concordance (F1 score = 0.95). Gene expression levels were highly correlated between blood and tumor tissue (R: 0.71-0.83). In the diagnostic cohort (n = 30 controls, n = 87 non-NET controls, n = 50 NET), NETest was significantly lower (p<0.0001) in controls (11±6.5) and non-NET controls (13±18) than NETs (61±31). The AUROCs were 0.93-0.97 and the diagnostic accuracy was 90-97.5%. As a diagnostic, the PSP-NETest was significantly better than CgA (accuracy: 56%, p<0.0001). For clinical samples, the PSP generated robust and accurate (>96%) scores and was significantly better (p<0.0001) than CgA. The assay protocol was consistent (r: 0.97) and reproducible (co-efficient of variation: 1.3-4.2%) across the two facilities.
CONCLUSION
The PSP protocol for the NETest has been established and prospectively tested in clinical samples. It is highly reproducible, has similar metrics (CV, categorization by control or NET) to the standard PCR assay and generates clinically concordant (>96%) NETest results. Moreover, it functions significantly more accurately than CgA.
Topics: Algorithms; Biomarkers, Tumor; Case-Control Studies; Chromogranin A; Gene Expression; Humans; Neuroendocrine Tumors; Pilot Projects; Polymerase Chain Reaction; Prospective Studies; RNA; Reproducibility of Results
PubMed: 31247038
DOI: 10.1371/journal.pone.0218592 -
Biotechnology and Bioengineering May 2021Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol...
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
Topics: African Swine Fever Virus; Animals; CRISPR-Cas Systems; Humans; Polymerase Chain Reaction; RNA, Guide, CRISPR-Cas Systems; RNA, Viral; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Swine
PubMed: 33615437
DOI: 10.1002/bit.27718 -
Journal of Visualized Experiments : JoVE Dec 2017In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection...
In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life. The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI. We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.
Topics: Arthroplasty; Humans; Polymerase Chain Reaction; Prostheses and Implants; Prosthesis-Related Infections; Sonication
PubMed: 29286460
DOI: 10.3791/55147 -
Advanced Biosystems Dec 2020There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding...
There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.
Topics: Animals; Cell Line; Equipment Design; Extracellular Vesicles; Humans; Mice; Microfluidic Analytical Techniques; Polymerase Chain Reaction; Single-Cell Analysis
PubMed: 33274611
DOI: 10.1002/adbi.201900307 -
The Journal of Veterinary Medical... Dec 2021A male Holstein-Friesian calf was born with multiple, cauliflower-like, pale pink cutaneous masses on the head and limbs. On histopathological examination, the cutaneous...
A male Holstein-Friesian calf was born with multiple, cauliflower-like, pale pink cutaneous masses on the head and limbs. On histopathological examination, the cutaneous masses were diagnosed as congenital cutaneous fibropapillomatosis. Those lesions involved focal proliferation of sebaceous gland in the dermis. There were no histological findings to suggest bovine papillomavirus infection, such as the presence of intranuclear inclusion bodies, large keratohyalin granules, and koilocytosis. Furthermore, papillomaviral antigens and DNA were not detected by immunohistochemistry and polymerase chain reaction, respectively. These results suggested that there was no association between these cutaneous lesions and bovine papillomavirus infection, and the lesions were considered as harmartomatous changes.
Topics: Animals; Immunohistochemistry; Male; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Skin
PubMed: 34732611
DOI: 10.1292/jvms.21-0503 -
Molecules (Basel, Switzerland) Jun 2023Polymerase chain reaction (PCR) has extensive bioanalytical applications in molecular diagnostics and genomic research studies for rapid detection and precise genomic...
Polymerase chain reaction (PCR) has extensive bioanalytical applications in molecular diagnostics and genomic research studies for rapid detection and precise genomic amplification. Routine integrations for analytical workflow indicate certain limitations, including low specificity, efficiency, and sensitivity in conventional PCR, particularly towards amplifying high guanine-cytosine (GC) content. Further, there are many ways to enhance the reaction, for example, using different PCR strategies such as hot-start/touchdown PCR or adding some special modifications or additives such as organic solvents or compatible solutes, which can improve PCR yield. Due to the widespread use of bismuth-based materials in biomedicine, which have not yet been used for PCR optimization, this attracts our attention. In this study, two bismuth-based materials that are inexpensive and readily available were used to optimize GC-rich PCR. The results demonstrated that ammonium bismuth citrate and bismuth subcarbonate effectively enhanced PCR amplification of the GNAS1 promoter region (∼84% GC) and APOE (75.5% GC) gene of Homo sapiens mediated by Ex Taq DNA polymerase within the appropriate concentration range. Combining DMSO and glycerol additives was critical in obtaining the target amplicons. Thus, the solvents mixed with 3% DMSO and 5% glycerol were used in bismuth-based materials. That allowed for better dispersion of bismuth subcarbonate. As for the enhanced mechanisms, the surface interaction of PCR components, including Taq polymerase, primer, and products with bismuth-based materials, was maybe the main reason. The addition of materials can reduce the melting temperature (T), adsorb polymerase and modulate the amount of active polymerase in PCR, facilize the dissociation of DNA products, and enhance the specificity and efficiency of PCR. This work provided a class of candidate enhancers for PCR, deepened our understanding of the enhancement mechanisms of PCR, and also explored a new application field for bismuth-based materials.
Topics: Humans; Dimethyl Sulfoxide; Glycerol; Bismuth; Solvents; Polymerase Chain Reaction
PubMed: 37298991
DOI: 10.3390/molecules28114515 -
Scientific Reports Sep 2019Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of...
Analogous to a photocopier, we developed a DNA microarray copy technique and were able to copy patterned original DNA microarrays. With this process the appearance of the copied DNA microarray can also be altered compared to the original by producing copies of different resolutions. As a homage to the very first photocopy made by Chester Charlson and Otto Kornei, we performed a lookalike DNA microarray copy exactly 80 years later. Those copies were also used for label-free real-time kinetic binding assays of apo-dCas9 to double stranded DNA and of thrombin to single stranded DNA. Since each DNA microarray copy was made with only 5 µl of spPCR mix, the whole process is cost-efficient. Hence, our DNA microarray copier has a great potential for becoming a standard lab tool.
Topics: Costs and Cost Analysis; DNA Probes; DNA, Single-Stranded; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Thrombin
PubMed: 31558745
DOI: 10.1038/s41598-019-50371-1