Polymerase Chain Reaction Strategy Research Articles

Cost of genetic testing, delayed care, and suboptimal treatment associated with polymerase chain reaction versus next-generation sequencing biomarker testing for genomic alterations in metastatic non-small cell lung cancer

Aims To assess US payers’ per-patient cost of testing associated with next-generation sequencing (NGS) versus polymerase chain reaction (PCR) biomarker testing strategies among patients with metastatic non-small cell lung cancer (mNSCLC), including costs of testing, delayed care, and suboptimal treatment initiation. Methods A decision tree model considered biomarker testing for genomic alterations using either NGS, sequential PCR testing, or hotspot panel PCR testing. Literature-based model inputs included time-to-test results, costs for testing/medical care, costs of delaying care, costs of immunotherapy [IO]/chemotherapy [CTX] initiation prior to receiving test results, and costs of suboptimal treatment initiation after test results (i.e. costs of first-line IO/CTX in patients with actionable mutations that were undetected by PCR that would have been identified with NGS). The proportion of patients testing positive for a targetable alteration, time to appropriate therapy initiation, and per-patient costs were estimated for NGS and PCR strategies combined. Results In a modeled cohort of 1,000,000 members (25% Medicare, 75% commercial), an estimated 1,119 had mNSCLC and received testing. The proportion of patients testing positive for a targetable alteration was 45.9% for NGS and 40.0% for PCR testing. Mean per-patient costs were lowest for NGS ($8,866) compared to PCR ($18,246), with lower delayed care costs of $1,301 for NGS compared to $3,228 for PCR, and lower costs of IO/CTX initiation prior to receiving test results (NGS: $2,298; PCR:$5,991). Cost savings, reaching $10,496,220 at the 1,000,000-member plan level, were driven by more rapid treatment with appropriate therapy for patients tested with NGS (2.1 weeks) compared to PCR strategies (5.2 weeks). Limitations Model inputs/assumptions were based on published literature or expert opinion. Conclusions NGS testing was associated with greater cost savings versus PCR, driven by more rapid results, shorter time to appropriate therapy initiation, and minimized use of inappropriate therapies while awaiting and after test results.

Cost-effectiveness analysis of diagnostic strategies for COVID-19 in Iran

BackgroundSince 2020, COVID-19 has become a global public health issue and has caused problems worldwide. This infection can lead to a fever and respiratory problems. Asymptomatic carriers of the virus are a significant part of the spread of the disease, so early screening and diagnosis of suspected cases of COVID-19 are essential. Generally, standard diagnostic methods include lung imaging (CT), polymerase chain reaction (PCR), and corona antibody (IgM&IgG) testing. However, the costs of the above tests for the healthcare system cannot be ignored, and evaluating the incremental costs against the additional benefit is necessary. Therefore, this study aimed to determine the cost-effectiveness of diagnostic methods for COVID-19 patients.Materials and methodsIn this research, an economic evaluation analysis was conducted to reveal the cost-effectiveness of the diagnostic strategies for COVID-19 from the service provider’s perspective. Basic information about the costs of CT, serology (IgG&IgM), and molecular (PCR) tests were collected from the Ministry of Health of Iran. The effectiveness data were calculated according to the sensitivity and specificity of the diagnostic tests for COVID-19. In this study, the incremental cost-effectiveness ratio (ICER) of the diagnostic strategies for COVID-19 was estimated, and the most cost-effective diagnostic strategy was determined. In calculating ICER and analyzing the sensitivity of the results, Treeage software was used.ResultsAccording to the calculated incremental effectiveness cost ratio for scenarios with 5, 10, and 50% prevalence of COVID-19 and according to the threshold defined by the World Health Organization, in the study, PCR, PCR, and IgG&IgM strategies are the most cost-effective diagnostic methods of the corona. Also, the results were not sensitive to the desired parameters based on the results of one-way sensitivity analysis.ConclusionNowadays there are various tests with different levels of accuracy in the diagnosis of COVID-19. In general, PCR tests are more cost-effective for low prevalence of Covid-19, while IgM&IgG tests are more cost-effective for high estimated prevalence. The results of this research can help policymakers and health system managers to validate the most accurate diagnostic method for COVID-19, considering the prevalence of the disease.

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR.

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 (Tm), 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.

Cost of genetic testing, delayed care, and suboptimal treatment associated with polymerase chain reaction (PCR) versus next-generation sequencing (NGS) testing strategies in metastatic non-small cell lung cancer (mNSCLC).

6638 Background: NGS testing for genomic alterations has been found to expedite time to appropriate targeted therapy initiation compared to PCR testing in mNSCLC. This study assessed US payers’ costs of testing, delayed care, and suboptimal treatment initiation for PCR versus NGS testing in patients with mNSCLC. Methods: A decision tree model evaluated testing for guideline-recommended alterations (EGFR, ALK, ROS1, BRAF, KRAS, MET, HER2, RET, NTRK1/2/3) in patients with newly-diagnosed mNSCLC from first test until appropriate therapy initiation using 1) liquid or tissue NGS (60% [90% tissue, 10% liquid] of patients), 2) exclusionary KRAS test followed by sequential PCR tests (5%), 3) sequential PCR tests (5%), or 4) hotspot panel (30%) PCR tests. The proportion of patients testing positive for a targetable alteration and time to appropriate therapy initiation were evaluated. Cost components included genetic testing, testing-related medical services, delayed care (i.e., costs for medical services while waiting for test results), immunotherapy [IO]/chemotherapy [CTX] initiation prior to test results, and suboptimal treatment initiation after test results (i.e., costs of first-line IO/CTX in patients falsely testing negative for an actionable mutation). Results: In a modeled cohort of 1 million members (75% commercial, 25% Medicare), an estimated 1,119 patients had mNSCLC and were tested. The proportion of patients testing positive for a targetable alteration was 45.9% for NGS and 39.4% for PCR testing (Table). Total mean per patient costs were lower for NGS ($8,866) than for PCR ($18,252), driven by more rapid treatment with appropriate therapy (2.1 versus 5.2 weeks), resulting in lower delayed care costs and IO/CTX costs prior to test results. Conclusions: Results suggest that NGS testing may lead to better patient care and reduced costs to the health system relative to PCR testing. Costs of inappropriate IO/CTX initiation are an important component of increased costs of PCR-based approaches. [Table: see text]

Photothermal Responsive Digital Polymerase Chain Reaction Resolving Exosomal microRNAs Expression in Liver Cancer.

Exosomal microRNAs have been studied as a good source of noninvasive biomarkers due to their functions in genetic exchange between cells and have been already well documented in many biological activities; however, inaccuracy remains a key challenge for liver cancer surveillance. Herein, a versatile duplex photothermal digital polymerase chain reaction (PCR) strategy combined with a lipid nanoparticle-based exosome capture approach is proposed to profile microRNAs expression through a 3-h easy-to-operate process. The microfluidically-generated molybdenum disulfide-nanocomposite-doped gelatin microcarriers display attractive properties as a 2-4°C s-1 ramping-up rate triggered by near-infrared and reversible sol-gel transforming in step with PCR activation. To achieve PCR thermocycling, the corresponding irradiation coordinating with fan cooling are automatically performed via a homemade control module with programs. Thus, taking the multiplexing capability of dual-color labeling, 19-31 folds higher in exosomal microRNA-200b-3p and microRNA-21-5p, and tenfold lower in microRNA-22-3p expressions relative to the control microRNA-26a-5p are quantified in two liver cancer cells (Huh7 and HepG2) than in those from the healthy cells. It is believed that this exosomal microRNA genotyping method would be highly applicable for liver cancer diagnostics.

Quantitative Analysis of the Effectiveness of Antigen- and Polymerase Chain Reaction-Based Combination Strategies for Containing COVID-19 Transmission in a Simulated Community

The number of coronavirus disease 2019 (COVID-19) cases continues to surge, overwhelming healthcare systems and causing excess mortality in many countries. Testing of infectious populations remains a key strategy to contain the COVID-19 outbreak, delay the exponential spread of the disease, and flatten the epidemic curve. Using the Omicron variant outbreak as a background, this study aimed to evaluate the effectiveness of testing strategies with different test combinations and frequencies, analyze the factors associated with testing effectiveness, and optimize testing strategies based on these influencing factors. We developed a stochastic, agent-based, discrete-time susceptible–latent–infectious–recovered model simulating a community to estimate the association between three levels of testing strategies and COVID-19 transmission. Antigen testing and its combination strategies were more efficient than polymerase chain reaction (PCR)-related strategies. Antigen testing also showed better performance in reducing the demand for hospital beds and intensive care unit beds. The delay in the turnaround time of test results had a more significant impact on the efficiency of the testing strategy compared to the detection limit of viral load and detection-related contacts. The main advantage of antigen testing strategies is the short turnaround time, which is also a critical factor to be optimized to improve PCR strategies. After modifying the turnaround time, the strategies with less frequent testing were comparable to daily testing. The choice of testing strategy requires consideration of containment goals, test efficacy, community prevalence, and economic factors. This study provides evidence for the selection and optimization of testing strategies in the post-pandemic era and provides guidance for optimizing healthcare resources.

Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning.

Megaprimer-based polymerase chain reaction (PCR) strategies allow the versatile and fast assembly and amplification of a myriad of tailor-made or random DNA sequences readily available for conventional or restriction-free (RF) cloning.In this chapter, we present a megaprimer-based PCR protocol that enables the expeditious construction of customized fusion genes ready for cloning into commercial expression plasmids. With the expanding use of protein tag technology in the most diverse application fields, this protocol remains a versatile and affordable solution for the synthesis and fusion of peptide tags/domains of interest.

An accurate genetic assay to identify human neutrophil antigen 2 deficiency.

We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans. HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now. A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T. In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null. The novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency.

Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products

Background aimsVector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct. MethodsThe authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer–probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct. ResultsddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors’ assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities. ConclusionsThe authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test–retest reliability, and that specific assays can be developed for detection of unique constructs.

COVID Detection using Symptoms and X-Ray

Abstract: The irritation established by the Covid sickness has caused a lot of destruction all around the world. Extremely fast dispersal of Covid has gagged just as imperiled the human development. Different cycles of research have focused on the clinical inclination for the finding of Corona Virus illness. Detection of covid prior of becoming dangerous is important but with the growing numbers it becomes difficult to manage it. Antigen and PCR tests which is reverse Transcription Polymerase Chain Reaction (RT-PCR) strategies for looking at the sickness have assumed a tremendous part in deciding the illness since the lively rise of COVID-19. The transmission of different freaks created in these two years has put a few inquiries on the accuracy and precise consequence of the business as usual. Along these lines, different radiological techniques like Computed Tomography (CT sweep) and X-beam, and so forth are being executed for the spotting of sickness due to the more fastidious outcome. With less resources many people have to face bad consequences and because of this reason many people lost their lives there is an emergent requirement for a fast solution so that detection is faster hence faster treatment. The investigatory review in the given report is about the utilization of Artificial Intelligence in hypothesizing the Covid infection utilizing an appropriate model. The most extreme precision accomplished among man-made reasoning classifiers adjusted in the given report is almost all the way. Along with detecting chest infection using X-Rays an application is needed by the people across the world to get to know whether they have covid or not on the basis of symptoms, early stage covid detection could be done easily by studying the symptoms so a model is required to detect whether a person is infected or not based on the symptoms user will fill. Keywords: COVID-19, X-Ray, Classification, CNN, Logistic Regression, Symptoms.

Applications of Nanozymology in the Detection and Identification of Viral, Bacterial and Fungal Pathogens.

Nanozymes are synthetic nanoparticulate materials that mimic the biological activities of enzymes by virtue of their surface chemistry. Enzymes catalyze biological reactions with a very high degree of specificity. Examples include the horseradish peroxidase, lactate, glucose, and cholesterol oxidases. For this reason, many industrial uses of enzymes outside their natural environments have been developed. Similar to enzymes, many industrial applications of nanozymes have been developed and used. Unlike the enzymes, however, nanozymes are cost-effectively prepared, purified, stored, and reproducibly and repeatedly used for long periods of time. The detection and identification of pathogens is among some of the reported applications of nanozymes. Three of the methodologic milestones in the evolution of pathogen detection and identification include the incubation and growth, immunoassays and the polymerase chain reaction (PCR) strategies. Although advances in the history of pathogen detection and identification have given rise to novel methods and devices, these are still short of the response speed, accuracy and cost required for point-of-care use. Debuting recently, nanozymology offers significant improvements in the six methodological indicators that are proposed as being key in this review, including simplicity, sensitivity, speed of response, cost, reliability, and durability of the immunoassays and PCR strategies. This review will focus on the applications of nanozymes in the detection and identification of pathogens in samples obtained from foods, natural, and clinical sources. It will highlight the impact of nanozymes in the enzyme-linked immunosorbent and PCR strategies by discussing the mechanistic improvements and the role of the design and architecture of the nanozyme nanoconjugates. Because of their contribution to world health burden, the three most important pathogens that will be considered include viruses, bacteria and fungi. Although not quite seen as pathogens, the review will also consider the detection of cancer cells and helminth parasites. The review leaves very little doubt that nanozymology has introduced remarkable advances in enzyme-linked immunosorbent assays and PCR strategies for detecting these five classes of pathogens. However, a gap still exists in the application of nanozymes to detect and identify fungal pathogens directly, although indirect strategies in which nanozymes are used have been reported. From a mechanistic point of view, the nanozyme technology transfer to laboratory research methods in PCR and enzyme-linked immunosorbent assay studies, and the point-of-care devices such as electronic biosensors and lateral flow detection strips, that is currently taking place, is most likely to give rise to no small revolution in each of the six methodological indicators for pathogen detection and identification. While the evidence of widespread research reports, clinical trials and point-of-care device patents support this view, the gaps that still exist point to a need for more basic research studies to be conducted on the applications of nanozymology in pathogen detection and identification. The multidisciplinary nature of the research on the application of nanozymes in the detection and identification of pathogens requires chemists and physicists for the design, fabrication, and characterization of nanozymes; microbiologists for the design, testing and analysis of the methodologies, and clinicians or clinical researchers for the evaluation of the methodologies and devices in the clinic. Many reports have also implicated required skills in mathematical modelling, and electronic engineering. While the review will conclude with a synopsis of the impact of nanozymology on the detection and identification of viruses, bacteria, fungi, cancer cells, and helminths, it will also point out opportunities that exist in basic research as well as opportunities for innovation aimed at novel laboratory methodologies and devices. In this regard there is no doubt that there are numerous unexplored research areas in the application of nanozymes for the detection of pathogens. For example, most research on the applications of nanozymes for the detection and identification of fungi is so far limited only to the detection of mycotoxins and other chemical compounds associated with fungal infection. Therefore, there is scope for exploration of the application of nanozymes in the direct detection of fungi in foods, especially in the agricultural production thereof. Many fungal species found in seeds severely compromise their use by inactivating the germination thereof. Fungi also produce mycotoxins that can severely compromise the health of humans if consumed.

Total cost of testing for genomic alterations associated with next-generation sequencing versus polymerase chain reaction testing strategies among patients with metastatic non-small cell lung cancer

Background To assess the total cost of testing associated with next-generation sequencing (NGS) versus polymerase chain reaction (PCR) testing strategies among patients with metastatic non-small cell lung cancer (mNSCLC) from a Medicare and US commercial payer’s perspective. Materials and Methods A decision tree model considered testing for genomic alterations in EGFR, ALK, ROS1, BRAF, KRAS, MET, HER2, RET, NTRK1 among patients with newly diagnosed mNSCLC using (1) liquid or tissue biopsy NGS tests, (2) exclusionary mutation (KRAS) test followed by sequential PCR tests, (3) sequential PCR tests, or (4) hotspot panel PCR tests. The alteration test sequence followed clinical guideline recommendations. Inputs based on literature, expert opinion, or assumptions included prevalence of mNSCLC, proportion of patients using each testing strategy (50% NGS [90% tissue, 10% liquid], 10% exclusionary, 10% sequential, 30% hotspot), proportion testing positive for each genomic mutation, rebiopsy rates, and costs for testing and associated medical care. Time to appropriate targeted therapy initiation and total costs were calculated for NGS, each PCR testing strategy, and all PCR strategies combined. Results Among a hypothetical plan of 1,000,000 members (75% commercial, 25% Medicare), 1,119 patients were estimated to have mNSCLC and be eligible for testing. Estimated mean time to appropriate targeted therapy was 2 weeks for NGS and 6 weeks for PCR (sequential: 9 weeks, exclusionary: 8 weeks, hotspot: 3 weeks). Mean per patient costs were $4,932 for NGS and $6,605 for PCR (exclusionary: $5,563, sequential: $6,263, hotspot: $7,066). Per patient costs were higher from a commercial perspective (NGS: $6,225; PCR: $8,430) relative to Medicare (NGS: $2,099; PCR: $2,646); nevertheless, NGS was the least costly testing strategy across plan types. Conclusion NGS was associated with the fastest time to appropriate targeted therapy initiation and lowest total cost of testing compared to PCR testing strategies for newly diagnosed patients with mNSCLC.

Integration of Microbial Transformation Mechanism of Polyphosphate Accumulation and Sulfur Cycle in Subtropical Marine Mangrove Ecosystems with Spartina alterniflora Invasion.

Excessive phosphorus can lead to eutrophication in marine and coastal ecosystems. Sulfur metabolism-associated microorganisms stimulate biological phosphorous removal. However, the integrating co-biotransformation mechanism of phosphorus and sulfur in subtropical marine mangrove ecosystems with Spartina alterniflora invasion is poorly understood. In this study, an ecological model of the coupling biotransformation of sulfur and phosphorus is constructed using metagenomic analysis and quantitative polymerase chain reaction strategies. Phylogenetic analysis profiling, a distinctive microbiome with high frequencies of Gammaproteobacteria and Deltaproteobacteria, appears to be an adaptive characteristic of microbial structures in subtropical mangrove ecosystems. Functional analysis reveals that the levels of sulfate reduction, sulfur oxidation, and poly-phosphate (Poly-P) aggregation decrease with increasing depth. However, at depths of 25-50cm in the mangrove ecosystems with S. alterniflora invasion, the abundance of sulfate reduction genes, sulfur oxidation genes, and polyphosphate kinase (ppk) significantly increased. A strong positive correlation was found among ppk, sulfate reduction, sulfur oxidation, and sulfur metabolizing microorganisms, and the content of sulfide was significantly and positively correlated with the abundance of ppk. Further microbial identification suggested that Desulfobacterales, Anaerolineales, and Chromatiales potentially drove the coupling biotransformation of phosphorus and sulfur cycling. In particular, Desulfobacterales exhibited dominance in the microbial community structure. Our findings provided insights into the simultaneous co-biotransformation of phosphorus and sulfur bioconversions in subtropical marine mangrove ecosystems with S. alterniflora invasion.

Role of Nanomaterials in Screenprinted Electrochemical Biosensors for Detection of Covid-19 and Post-Covid Syndromes– a Metareview

Covid-19 still remains as global pandemic surging with different variants in various countries resulted into Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). An escalation in random testing for Covid-19 is considered as golden standard by WHO to control the spread of Corona virus. At present, the tests are performed to investigate the infection genetic materials or to detect the antibodies already produced by the immune system to invade the infection. Nucleic acid amplification tests (NAATs) and antigen tests are two viral based tests for detection of current virus and the serology test is antibody test for detection of past infection. Reverse transcriptase real-time polymerase chain reaction (RT-PCR) and nucleic acid hybridization strategies are the two highly sensitive outstanding tests used for the detection of SARS-CoV-2 in the DNA by the former and RNA/DNA by the later. Low controlled below -20oC temperature with a professional technician and time-intensive are the major drawbacks reported. Apart from the test for virus, antigen and antibodies several other alternative hematological tests like CRP: C-Reactive Protine, Ferritine, IL-6: Interleukin 6, Blood Platelets and WBC: White Blood Cells plasma levels became essential for the detection of Covid-19 and the post Covid complications. To overcome these limitations a rapid, efficient, low-cost and easy-to-implement test kits are in imperative need to meet the requirement. Electrochemical sensors are considered as potential, ultra-fast and point-of-care sensors used as self testing kits. In the present work, a meta review of various modified electrodes with different nanomaterials such as graphene, carbon nanotubes, and nano-dopants used as electrochemical sensors in association with testing various different parameters for detection of covid-19. A detailed discussion on the merits and demerits of present testing patterns and scope of screen-printed electrochemical sensors modified with different nanomaterials and their advantages are presented. Keywords: Corona Virus, Electrochemical Sensors, Nanomaterials modified electrodes, Ultra-fast, and Point-of-care Figure 1

In-house abbreviated qualification of a real-time polymerase chain reaction method and strategies to amplify mycoplasma detection in human mesenchymal stromal cells

Background aimsIn this study, the authors performed an in-house abbreviated qualification of a commercially available real-time polymerase chain reaction (PCR) kit for limit of detection (LOD), matrix interference and ruggedness of mycoplasma detection in a human bone marrow-derived mesenchymal stromal cell (MSC(M)) investigational cell product (NCT02351011). The approach used was similar to an abbreviated qualification the authors previously conducted for endpoint PCR, which was accepted by Canadian regulators for final product release of the same MSC(M) investigational cell product for treatment of osteoarthritis patients (NCT02351011). With patient consent, biobanked MSCs(M) were re-analyzed by real-time PCR for mycoplasma detection to conduct in-house qualification of the kit. MethodsLOD was determined by spiking MSCs(M) with a series of 10-fold dilutions of two commercially available genomic DNA (gDNA) reference standards for Mycoplasma arginini (M. arginini) and Mycoplasma hominis (M. hominis). Matrix interference was tested by using 10-fold dilutions of MSC(M)s down to 4500 cells/mL. Polyadenylic acid (poly[A]) was used to improve DNA recovery in samples with 4500–45 000 MSCs(M)/mL. Real-time PCR tests performed on different days were compared to evaluate ruggedness. ResultsReal-time PCR analysis showed a conservative LOD of 40 genome copies (GCs)/mL and 240 GCs/mL, which are equivalent to 10 colony-forming units (CFUs)/mL, for M. arginini and M. hominis, respectively. According to a less conservative manufacturer-based criterion for positivity, the kit detected 0.4 GC/mL (0.1 CFU/mL) and 24 GCs/mL (1 CFU/mL) M. arginini and M. hominis, respectively. Real-time PCR with different MSC(M) dilutions did not show matrix interference. However, DNA recovery was compromised at MSC(M) concentrations at or below 45 000 cells/mL. The addition of poly(A) as a DNA carrier improved DNA recovery and allowed an LOD, considered here to be equivalent to 10 CFUs/mL, to be achieved, which was not possible in diluted MSC(M) samples (≤45 000 cells/mL) in the absence of poly(A). Ruggedness was demonstrated with tests (n = 18) performed on different days, with an average overall inter-assay percent coefficient of variation of less than 4 for M. arginini (3.62 [400 GCs/mL], 3.61 [40 GCs/mL]) and less than 3 for M. hominis (2.83 [2400 GCs/mL], 1.95 [240 GCs/mL]). ConclusionsA commercially available real-time PCR mycoplasma detection kit was qualified for evaluating mycoplasma contamination in investigational MSC(M) products and met the criteria used previously (and accepted by Canadian regulators) for in-house qualification of an endpoint PCR mycoplasma detection kit, and the addition of poly(A) addressed the poor recovery of mycoplasma gDNA in samples with low cell numbers.

Performance of TaqMan probes for the detection of sexually transmitted infections in South African women.

Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis and Mycoplasma genitalium are the four main aetiologies of sexually transmitted infections responsible for vaginal discharge syndrome (VDS). Commercially available multiplex polymerase chain reaction (PCR) assays are expensive and generally not customisable. We evaluated a highly customisable singleplex PCR approach by testing it in parallel with the Anyplex™ II STI-7 detection assay in a cohort of South African women that presented with VDS between May 2016 and January 2017. Our multiple singleplex PCR strategy proved to be a simple, accurate, rapid, affordable and scalable option for diagnosing VDS.

Detection, Mapping, and Proteotyping of SARS-CoV-2 Coronavirus with High Resolution Mass Spectrometry.

A high resolution mass spectrometry approach has been applied for the first time to detect and characterize SARS-CoV-2 coronavirus in cell cultured and nasopharyngeal swab specimens. Peptide ions for three of the most abundant structural viral proteins (membrane, nucleocapid, and spike) are detected and assigned directly, by virtue of the high resolution and mass accuracy within the mass maps of whole virus digests, without the need for tandem mass spectrometry (MS/MS). MALDI-MS based approaches offer high sample throughput and speed, compared with those of LC–MS strategies, and detection limits at some 105 copies, or orders of magnitude less with selected ion monitoring, that compete favorably with conventional reverse transcription polymerase chain reaction (RT-PCR) strategies. The detection of signature peptides unique to SARS-CoV-2 coronavirus over those from the influenza virus allows for its unambiguous detection.

Outpatient Urinary-Tract-Infection-Like Symptoms: Causative Microbial Survey Utilizing Multiplex Quantitative Polymerase Chain Reaction Methodology

Urinary tract infections (UTIs) are among the most common maladies afflicting the human population globally. A wide variety of microbial pathogens are responsible for causing UTIs that are often recurrent, especially in the elderly population, resulting in an economic burden of billions of dollars annually. In this study, we present data from a large population of symptomatic UTI patients tested using a multiplexed, real time quantitative polymerase chain reaction (qRT-PCR) strategy. Our data demonstrate that instances of UTI increase with age for all the tested pathogens, with the exception of Mycoplasma spp. and Ureaplasma spp., which were found to be significantly higher in the younger population. Furthermore, the use of the qRT-PCR approach was also found to be effective in detecting polymicrobial UTIs. With the limitations of classical microbial culture techniques traditionally used for UTI diagnosis, we demonstrate that a rapid and comprehensive technique like RT-PCR can be an effective tool for detecting and managing UTIs.

Rapid and Efficient Live Zebrafish Embryo Genotyping.

Most current methods for genotyping zebrafish embryos require sacrifice or raising the animal to at least 1 month of age for fin amputation. These limitations restrict the use of zebrafish and increase time and costs for experiments. This article introduces an innovative method for genotyping live zebrafish embryos. The method utilizes enzyme to extract a small amount of genetic material from the skin tissue of the embryo. Then, using conventional polymerase chain reaction (PCR) strategy, the embryo is genotyped. This approach was successful >95% of the time while maintaining high viability (>90%) of the embryo. This effective method can facilitate high-throughput screening and other applications in zebrafish.

A Review on NanoPCR: History, Mechanism and Applications.

Polymerase Chain Reaction (PCR) is one of the most common technologies used in many laboratories to produce millions of copies of targeted nucleic acid under in vitro conditions. However, PCR faces multiple challenges including limited availability of DNA in the sample, high GC contents of the template, low efficiency, and specificity in amplification. Moreover, some DNA fragments are very difficult to amplify due to their secondary structure and high melting temperature requirement. To overcome these challenges, many approaches including the application of PCR additives in PCR mixture; change in instrument design; optimization of PCR system by using the accurate concentration of magnesium ions, primers, and cycle number; enzyme modification; and setting up the new touchdown and nested PCR strategies have been adopted. Although these approaches have enriched the output of PCR, they are not all-purpose and optimization can be case dependent. Nanometer-sized materials (nanomaterials) have offered a possible solution to these problems as these materials have exceptional physio-chemical properties as compared to macroscopic materials. Among these nanomaterials, silicon-based materials, carbon-based materials, semiconductor quantum dots (QDs), and some metals are well-known PCR enhancer. Hence, new PCR has been designed to utilize the unique properties of nanomaterial and is known as nanomaterial-assisted PCR or simply nanoPCR. Results of many studies have shown that the combination of these nanomaterials and biomolecules can mimic the DNA replication process successfully as present in the living organism. In this review, we have discussed the role of these different nanomaterials one by one and also discussed the mechanisms through which these nanomaterials enhance the efficiency of PCR.