Validity Of Assay Research Articles

Multicenter validation of an RNA-based assay to predict anti-PD-1 disease control in patients with recurrent or metastatic head and neck squamous cell carcinoma: the PREDAPT study.

Despite advances in cancer care and detection, >65% of patients with squamous cell cancer of the head and neck (HNSCC) will develop recurrent and/or metastatic disease. The prognosis for these patients is poor with a 5-year overall survival of 39%. Recent treatment advances in immunotherapy, including immune checkpoint inhibitors like pembrolizumab and nivolumab, have resulted in clinical benefit in a subset of patients. There is a critical clinical need to identify patients who benefit from these antiprogrammed cell death protein 1 (anti-PD-1) immune checkpoint inhibitors. Here, we report findings from a multicenter observational study, PREDicting immunotherapy efficacy from Analysis of Pre-treatment Tumor biopsies (PREDAPT), conducted across 17 US healthcare systems. PREDAPT aimed to validate OncoPrism-HNSCC, a clinical biomarker assay predictive of disease control in patients with recurrent or metastatic HNSCC treated with anti-PD-1 immune checkpoint inhibitors as a single agent (monotherapy) and in combination with chemotherapy (chemo-immunotherapy). The test used RNA-sequencing data and machine learning models to score each patient and place them into groups of low, medium, or high. The OncoPrism-HNSCC prediction significantly correlated with disease control in both the monotherapy cohort (n=62, p=0.004) and the chemo-immunotherapy cohort (n=50, p=0.01). OncoPrism-HNSCC also significantly predicted progression-free survival in both cohorts (p=0.015 and p=0.037, respectively). OncoPrism-HNSCC had more than threefold higher specificity than programmed death-ligand 1 combined positive score and nearly fourfold higher sensitivity than tumor mutational burden for predicting disease control. Here, we demonstrate the clinical validity of the OncoPrism-HNSCC assay in identifying patients with disease control in response to anti-PD-1 immune checkpoint inhibitors. NCT04510129.

Key performance evaluation of commercialized multiplex rRT-PCR kits for respiratory viruses: implications for application and optimization.

The complexity and severity of viral respiratory tract infections (RTIs) emphasize the pivotal role of precise diagnosis for viral RTIs in guiding effective public health responses and ensuring appropriate medical interventions, given the substantial population at risk. This study highlights the necessity and importance of evaluating the analytical validity of multiplex real-time reverse transcription polymerase chain reaction assays, offering valuable insights into their optimization and application.

B-341 Comparative Study of Free Light Chains in the Framework of the Project Prevalence of Monoclonal Gammopathies in Adult Uruguayan Population

Abstract Background Within the framework of a study to evaluate the Prevalence of Monoclonal Gammopathies in the Adult population of Uruguay through detection monoclonal bands by capillary electrophoresis and free light chains in serum, the aim is evaluating the comparison of quantification of FLC (Free Light Chains) in this group of patients with 2 assays: Freelite Biding Site® versus Sebia® FLC Elisa. Methods Study period: September 2021 to November 2022. Prospective, single-cohort, descriptive study. Approved by the Hospital de Clinicas Ethics committee, Montevideo, Uruguay.A total of 3905 patients were included (≥40 years, without previously known plasma cell disorder).Serum samples were collected from three health centers, and processed in Clinical Chemistry Laboratory at Hospital de Clinicas, following an algorithm designed to perform electrophoretic proteinogram (by capillary electrophoresis); suspicious samples with monoclonal bands were confirmed by immunotyping, and in some cases by immunofixation (difficult interpretation), and quantification FLC by 2 different assays (Sebia FLC: manual sandwich ELISA technique- BIO-TEK wash/reader; Freelite The Binding Site immunoturbidimetric assay : Spa Plus and Optilite instruments. Comparative analysis of FLC is a transversal and descriptive study.For statistical analysis: Spearman test, Passing and Bablok and Bland-Altman plot. Results κFLC, λFLC and ratio κ/ λ were measured in 93 of the 104 positive serum samples for Monoclonal Gammopathies (new diagnoses, prevalence 2.7%), using two groups Sebia FLC and Spa Plus Freelite, and Sebia FLC and Optilite Freelite. During the project, the Spa Plus (n=48) FLC was replaced with Optilite (n=45), since the comparative group was not analytically homogeneous, so the comparison was carried out in two groups. Spearman rank correlation coefficients between first group (N:48) for κFLC, λFLC and ratio κ/ λ were r=0.596, 0,549, 0.709, respectively (p<0.0001); for the second group (N:45) Spearman rank correlation coefficients were r=0.778, 0.714, 0.878, respectively (p<0.0001). The first group showed good Spearman rank correlation for ratio, although less better correlation for kappa and lambda isolated. Bland Altman showed high media differences in assays for values for Sebia. For the second group, there was a better Spearman Correlation for ratio and in kappa and lambda isolated too. Bland-Altman plot revealed, in the second group analysis, that Freelite provides higher values than Sebia in Kappa and ratio, but not in Lambda. Passing Bablock showed differences systematic and proportional in different group of comparatives. Conclusions There is a good agreement; despite this, within high FLC concentrations, there are particularly significant differences between both assays, in both groups. We found in same cases FLC quantifications with Freelite ratios lower than those reported by the Elisa Sebia assay, which in these patients could change the diagnosis and therefore the opportunity for treatment or the time between follow-ups. In other cases, greater amount of ratio FLC assayed by Freelite, which may represent loss of linearity in subsequent dilutions. Falsely high results may occur due to polymerization of light chains.Our results suggest that Sebia FLC is a valid assay alternative, but needs more studies to demonstrate these findings; the prospective follow up of this group promises more.

Confounding variables such as previous transplants, gender, ethnicity, and primary disease complicate the diagnostic validity of single-antigen bead-based non-HLA antibody assays

Confounding variables such as previous transplants, gender, ethnicity, and primary disease complicate the diagnostic validity of single-antigen bead-based non-HLA antibody assays

Comparison of Age-Related Decline and Behavioral Validity in C57BL/6 and CB6F1 Mice.

Variability in physical resilience to aging prompts a comprehensive examination of underlying mechanisms across organs and individuals. We conducted a detailed exploration of behavioral and physiological differences between C57BL/6 and CB6F1 mice across various age groups. In behavioral assays, B6 mice displayed superior performance in rotarod tasks but higher anxiety while CB6F1 mice exhibited a decline in short-term memory with age. Grip strength, long-term memory, and voluntary wheel running declined similarly with age in both strains. Examining physiological phenotypes, B6 mice exhibited lower body fat percentages across ages compared to CB6F1 mice, though cataract severity worsened with age in both strains. Analysis of cardiac functions revealed differences between strains, with worsening left ventricular hypertrophy and structural heart abnormalities with age in CB6F1 mice along with higher blood pressure than B6. Lesion scores showed an age-related increase in heart, kidney, and liver lesions in both strains, while lung lesions worsened with age only in CB6F1 mice. This study underscores the validity of behavioral assays and geropathology assessment in reflecting age-related decline and emphasizes the importance of considering strain specificity when using mouse models to study human aging.

Using QUASR-PCR as a field-based genotyping assay for a tick acaricide resistance marker

A novel, turnkey, field-based workflow was developed and validated using Rhipicephalus microplus DNA as a template to detect the presence of the voltage-gated sodium channel kdr mutation. The field-based compatible workflow comprises manual sample homogenization for DNA extraction, PCR amplification of the targets in a closed tube, and end-point detection of the PCR products. An R. microplus species-specific assay was also included to confirm species identity and ensure the validity of the kdr mutation assay. The assays were sensitive and specific to the targets, and the workflow resulted in a turnaround time of approximately 1 h at a low cost. The novel combination of PCR with closed-tube and end-point fluorescent detection allows for easy conversion of existing conventional lab-based PCR assays into field-based detection assays. The incorporation of custom-designed 3D-printed components in the workflow provides easy adaptability and modification of the components for diverse nucleic acid detection workflows.

Exploring ultrasonic communication in mice treated with Cannabis sativa oil: Audio data processing and correlation study with different behaviours.

Studying ultrasonic vocalizations (USVs) plays a crucial role in understanding animal communication, particularly in the field of ethology and neuropharmacology. Communication is associated with social behaviour; so, USVs study is a valid assay in behavioural readout and monitoring in this context. This paper delved into an investigation of ultrasonic communication in mice treated with Cannabis sativa oil (CS mice), which has been demonstrated having a prosocial effect on behaviour of mice, versus control mice (vehicle-treated, VH mice). To conduct this study, we created a dataset by recording audio-video files and annotating the duration of time that test mice spent engaging in social activities, along with categorizing the types of emitted USVs. The analysis encompassed the frequency of individual sounds as well as more complex sequences of consecutive syllables (patterns). The primary goal was to examine the extent and nature of diversity in ultrasonic communication patterns emitted by these two groups of mice. As a result, we observed statistically significant differences for each considered pattern length between the two groups of mice. Additionally, the study extended its research by considering specific behaviours, aiming to ascertain whether dissimilarities in ultrasonic communication between CS and VH mice are more pronounced or subtle within distinct behavioural contexts. Our findings suggest that while there is variation in USV communication between the two groups of mice, the degree of this diversity may vary depending on the specific behaviour being observed.

Abstract 2490: Quantification of tumor fraction and outcomes association in a real-world non-small cell lung cancer (NSCLC) cohort using a tissue agnostic epigenomic circulating tumor DNA (ctDNA) assay

Abstract Background: The validity of ctDNA assays for evaluating molecular response to therapy by measuring changes in variant allele fraction (VAF) while on treatment is well established, but they are prone to limitations such as low ctDNA levels and interference from copy number variation and clonal hematopoeisis (CH), which may be overcome by methylation-based quantification. ctDNA levels may be monitored throughout a patient's (pts) journey to indicate when relapse or progression is present, often sooner than current methods (RECIST). Here we describe the performance of a methylation-based ctDNA assay to quantify ctDNA levels and correlate changes with outcomes in a real-world NSCLC cohort. Methods: RADIOHEAD is a pan-tumor observational study of 1200 patients on standard of care ICI treatment regimens with plasma samples collected prospectively for retrospective analysis. We randomly selected 241 NSCLC pts that have a combined total of 761 samples (236 baseline, 191 C3D1, and 128 at 6 months post first dose). Samples were analyzed using a commercial NGS assay, which quantifies circulating tumor fraction (cTF) via interrogating thousands of methylation sites from ctDNA, validated with LoB, LoD, LoQ, and linearity studies. Cox proportional hazards (CPH) were used for comparison of real-world progression free survival (rwPFS). Gender, age, disease stage, and blood-assessed tumor mutational burden (bTMB) were included as covariates. Median rwPFS was calculated using Kaplan Meier analysis. rwPFS ordinal groups were defined as PFS event within 3mo, 3-6mo, 6-12mo, and greater than 12mo, and association with early ctDNA changes was assessed with a chi-squared test. Results: In this cohort, 4, 11, 83 and 142 pts were stage I to IV, respectively (1 unknown). Methylation based detection of ctDNA at baseline or C3D1 was associated with shorter rwPFS (baseline: mPFS 12.7 mo [9.3-19.5] vs NR [16.4-NR]; HR=2.2 [1.3-3.7] p<0.005, C3D1: mPFS 12.4 mo [9.3-16.1] vs NR [19.5-NR] HR= 2.2 [1.3-3.5], p<0.005) independent of stage, gender, or age. Longer rwPFS was associated with >95% reduction in cTF from baseline to C3D1 or low ctDNA at both timepoints (rwPFS <3mo = 12.5%, 3-6 mo = 30.8%, 6-12 = 37.1%, ≥12 mo = 55.3%; N=153, p=0.015). For pts with ctDNA not detected at C3D1, ctDNA detection at 6 months post first dose was strongly associated with shorter rwPFS (mPFS 13.5 mo [9.5-NR] vs. NR [19.5-NR]; HR= 6.2 [1.9-20.1], p<0.005). Conclusions: These data demonstrate a significant association of ctDNA detection via methylation-based cTF and on-treatment changes in cTF with rwPFS. Furthermore, subsequent cTF detection 6-months on-treatment, without cTF detected early on-treatment was associated with worse outcomes, suggesting the value of serial monitoring to improve identification of patients likely to progress. Citation Format: Sara Wienke, Sean Gordon, Samantha Liang, Jing Wang, Reagan Barnett, Kyle Chang, Shile Zhang, Carin R. Espenschied, Katie Quinn, Kimberly Banks. Quantification of tumor fraction and outcomes association in a real-world non-small cell lung cancer (NSCLC) cohort using a tissue agnostic epigenomic circulating tumor DNA (ctDNA) assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2490.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs.

Multiparametric flow cytometry (MFC) represents an essential tool for immune monitoring, and validation of MFC panels is a fundamental prerequisite in routine laboratory settings as well as for translational and clinical research purposes. Regulatory T cells (TREGs) constitute a subset of CD4+ effector T cells that modulate the immune response in numerous settings, including autoimmune disease, allergy, microbial infection, tumor immunity, transplantation, and more. These cells comprise a small fraction of total CD4+ T cells in human peripheral blood and mouse spleen. In oncology, TREG cells are highly relevant, as they are involved in the suppression of the anti-tumor response in many types of cancer, to the extent that the first immune checkpoint inhibitor approved for clinical use in humans was a monoclonal antibody directed against CTLA-4, a molecule functionally associated with TREGs. Due to all these factors, robust assays are mandatory to accurately determine TREG cell frequency and function. Here, we describe the validation of an 8-color flow-cytometry protocol for TREG detection and analysis in a real-world laboratory scenario. The entire process includes the workflow plan and the standard operating procedure resembling each phase, from the panel design to the staining, acquisition, and analysis steps. Validation is planned to be performed in replicates on fresh whole blood samples derived from multiple healthy subjects. The analytical validity of the TREG cell assay is ensured by testing the intra-assay accuracy. The detailed procedure for the entire process is accompanied by important troubleshooting suggestions and other useful tips.

PTau181 plasma biomarker performance as an inclusion criterion for Alzheimer’s Disease clinical trials

AbstractBackgroundAdvances in ultrasensitive detection techniques for blood biomarkers allow the quantification of AD‐specific phosphorylated Tau proteins, including Tau phosphorylated at threonine 181 (pTau181) in patient plasma. pTau blood‐based biomarker have shown great promise as inclusion criteria and secondary endpoint evaluation in clinical trials.MethodThe University of British Columbia (UBC) CARD biobank plasma samples from clinically diagnosed AD and non‐AD patients were used to establish clinical and analytical validity of the pTau181 plasma assay per FDA fit‐for‐purpose guildelines (Neurcode USA, Inc.). We assessed the analytical measurement interval, clinical reportable range, linearity, intra‐laboratory precision, specimen stability, interference, and clinical performance.ResultThe pTau181 plasma assay provides a robust and accurate biomarker approach for independent determination of AD, with an AUC of 0.9 in our validation study cohort. The cut‐point (≥ 30 ng/L) had 100% sensitivity and 88% specificity for AD diagnosis in autopsy‐confirmed samples. The plasma pTau181 assay appears to be performing well as additional screening metric for inclusion of mild‐to‐moderate AD subjects in Phase 3 clinical trials (i.e. RETHINK‐ALZ and REFOCUS‐ALZ). 89% of the clinical sites participating in the REFOCUS‐ALZ study and 80% of sites participating in the RETHINK‐ALZ had at least 70% of screened subjects meeting this criterion.ConclusionAD plasma biomarker (pTau181) quantified using an RUO assay has great potential both as a diagnostic tool and to streamline clinical trials in AD.

SY12-1 Assessment on clinical validity of PD-L1 assays in lung cancer

SY12-1 Assessment on clinical validity of PD-L1 assays in lung cancer

Utility of glutaraldehyde-fixed turkey red blood cells for influenza virus detection after 18 months of storage

Turkey red blood cells (tRBCs) are an essential reagent used in the laboratory diagnosis of influenza viruses. Fresh tRBCs when stored at 4 °C have a shelf life of less than a week. Previous studies have shown the utility of glutaraldehyde-fixed tRBCs, with an increased shelf life, for use in hemagglutination (HA) assays. In the present study, we report their functionality after storage for 18 months, at -80 °C. Three influenza A subtypes, namely, H3N2, H1N1 and H5N1, were used in the study. Hemagglutination assay was performed using freshly prepared 0.5 % tRBCs suspension and stored 1 % glutaraldehyde-fixed tRBCs. There was no significant difference in the HA titers obtained using fresh and stored tRBCs. The validation of the HA assay was carried out, to determine the specificity, linearity, precision, accuracy, and robustness of the assay. All of the titers were within the acceptable range, indicating the validity of the HA assay using stored tRBCs. Hemagglutination inhibition assay was also performed to compare the antibody titers obtained using stored and fresh tRBCs. The stored RBCs also gave equivalent antibody titers, as compared to the fresh tRBCs. Thus, the present study demonstrates the utility of glutaraldehyde-fixed tRBCs after one and a half years of storage.

Dense fine speckled nuclear immunofluorescence: A mildly reassuring antinuclear antibody pattern meriting consideration.

Antinuclear antibodies (ANAs) are regarded as a hallmark of connective tissue diseases (CTDs) and play a key role in their diagnosis, but the value of some particular antibodies in management of patients and the disease prognosis is controversial. The mechanism underlying the production of ANAs in CTDs, other chronic inflammatory conditions and even in healthy people, is not completely elucidated. Anti-DFS70 antibodies connected with the dense fine speckled autoantigen of 70 kD, known as the lens epithelium-derived growth factor p75, are a subgroup of ANAs. Their presence and coexistence with other antibodies and their clinical significance are the matter of debate. Based on literature data, the authors focused on current knowledge explaining the role of anti-DFS70 antibodies in selected CTDs. However, the literature data is ambiguous and does not fully support the validity of the anti-DFS70 assay for a specific CTD diagnosis. Most researchers claim that the presence of anti-DFS70 as the only one usually exclude the diagnosis of CTD. Nevertheless, its coexistence with other ANAs is not an excluding factor but has predictive value due to more favorable course of CTD. Such situations may also suggest an enhanced risk of the development of a CTD in the future. Although more studies are needed in this field, it seems reasonable to ascertain the presence of anti-DFS70 in routine clinical practice.

Diagnostic validity of molecular diagnostic assays for white spot syndrome virus at different severity grades

The high virulence of the white spot syndrome virus (WSSV) in acute infections and the lack of effective treatments underscore the necessity for a rapid, accurate, and efficient diagnostic process to control white spot disease. The analytical sensitivity of diagnostic assays (polymerase chain reactions; PCRs and rapid diagnostic kit) at different severity grades of WSSV infection were determined using the 95% limit of detection (LOD95%). The LOD95% of nested, real-time, and one-step PCRs and rapid diagnostic kit were 0.70, 4.67, 1.19, and 79,434.65 viral genome copies/reactions, respectively. From the intramuscular challenge tests conducted under different dose and temperature conditions, the WSSV severity grades of time-course collected whiteleg shrimp (Litopenaeus vannamei) and dead or live shrimps were determined based on the viral loads of the pleopod. By applying the WSSV severity grades, a total of 92 shrimps were classified as G0. Furthermore, 92, 66, 199, and 79 shrimps were classified as G1, G2, G3, and G4, respectively. Additionally, 222 shrimps were classified as negative as WSSV was not confirmed in the nested PCR assay. Diagnostic sensitivity (DSe) and specificity (DSp) values of molecular diagnostic assays were compared with those of nested PCR in artificial WSSV-infected shrimp to evaluate diagnostic performance. The real-time and one-step PCRs exhibited a DSe >92.4% for G0 grade (approximately 101–102 copies/mg), indicating WSSV detection at low copy numbers. The rapid diagnostic kit presented a DSe >92.4% for G2 grade (approximately 104–105 copies/mg), suggesting the detection of WSSV-infected shrimp with clinical signs during the endemic period. These results suggest that the strategy of presumptive diagnosis using one-step PCR and rapid diagnostic kit at different seasonal periods followed by confirmatory diagnosis using real-time PCR assay could aid in controlling WSD with rapidity, accuracy, and cost-effectiveness.

Immunogenicity and safety of a pentavalent meningococcal ABCWY vaccine in adolescents and young adults: an observer-blind, active-controlled, randomised trial

Immunogenicity and safety of a pentavalent meningococcal ABCWY vaccine in adolescents and young adults: an observer-blind, active-controlled, randomised trial

Synthesis of molecularly imprinted polymers based on nitrogen-doped carbon dots for specific detection of chlortetracycline by reversed phase microemulsion method

Among the tetracycline antibiotics, chlortetracycline (CTC) is the most frequently used antibiotic except for tetracycline (TC) for enhancing the ability of the organism to fight bacterial infections. The poor metabolism and degradability of CTC can cause serious health effects. Most studies have focused on the detection and analysis of TC, and research on CTC is relatively scarce. This is because the structures of CTC and TC and oxytetracycline (OTC) are extremely similar, and even indistinguishable. In this study, CTC was used as a template molecule and a molecularly imprinted layer was coated on the surface of highly fluorescent N-CDs using a reversed-phase microemulsion method to form N-CDs@MIPs. It was possible to specifically identify CTC without the influence of TC and OTC, which are extremely similar in structure. By comparing with the non-imprinted polymer (N-CDs@NIPs), it exhibited high sensitivity and selectivity with an imprinting factor of 2.02. And it was used in the determination of CTC in milk with recoveries and relative standard deviations of 96.7%–109.8% and 0.64%–3.27%, respectively, with high accuracy and precision. The specificity of the measurement is excellent compared with other assays, and it is a valid and reliable assay.

QPCR Analysis of Quorum Sensing Genes of Pseudomonas aeruginosa

A well-designed and properly validated primer produces a specific and efficient qPCR assay. The primer’s melting temperature, G-C content, its size, as well as the amplicon’s size are the principal considerations while designing the primer for this study and it was in accordance with the MIQE guidelines. Subsequently, the designed primer was evaluated prior to the analysis to verify its efficiency and precision for the relative quantification of the gene transcription analysis. The validity of a qPCR assay is reliant on three elements; the qPCR efficiency (E) and the R2 as well as the slope, that were derived from constructed standard curve. The resulted E for the genes lasI, lasR, rhlI, rhlR and rplS are 92%, 93%, 96%, 92% and 94%, respectively. While the R2 and the slope value for these genes are 0.9991 and -3.523 for the lasI, 0.9991 and -3.501 for the lasR, 0.9989 and -3.434 for the rhlI, 0.9999 and -3.535 for the rhlR, and 0.9935 and -3.487 for the rplS. Melt curve analyses carried out post qPCR assay resulted in amplification of a single product. Resulted E, R2 and slope for all studied genes fell between the acceptable range, validating the use of designed primer for further analysis in the changes in transcription level of quorum sensing genes in treated Pseudomonas aeruginosa.