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UNLABELLED: Given the challenges that SARS-CoV-2 variants have caused in terms of rapid spread and reduced vaccine efficacy, a rapid and cost-effective assay that can detect new and emerging variants is greatly needed worldwide. We have successfully applied the xenonucleic acid-based molecular-clamping technology to develop a multiplex reverse-transcription quantitative real-time PCR assay for SARS-CoV-2 multivariant detection. The assay was used to test 649 nasopharyngeal swab samples that were collected for clinical diagnosis or surveillance. The assay was able to correctly identify all 36 Delta variant samples as it accurately detected the D614G, T478K, and L452R mutations. In addition, the assay was able to correctly identify all 34 Omicron samples by detecting the K417N, T478K, N501Y, and D614G mutations. This technique reliably detects a variety of variants and has an analytical sensitivity of 100 copies/mL. In conclusion, this novel assay can serve as a rapid and cost-effective tool to facilitate large-scale detection of SARS-CoV-2 variants. IMPORTANCE: We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

OBJECTIVES: Patients with VEXAS syndrome carry mutations of UBA1 gene coding for the E1 enzyme. The three most frequent mutations are p.M41T(122T > C), p.M41V (c.121A > G), and p.M41L (c.121A > C) in codon 41 of exon 3. Currently, sanger sequencing was mainly used to detect these mutations, which has low sensitivity and low throughput. There is a need of high sensitivity, simple and high throughput method to characterize patients with VEXAS syndrome. METHODS: Based on our proprietary XNA technology, we have developed a QClamp® Plex platform to detect eight mutations in a single reaction using the Luminex xMap technology. The assay sensitivity, specificity and precision were subsequently evaluated. Furthermore, the reference interval and clinical sensitivity/specificity were estimated using clinical healthy/positive DNA samples and the sanger sequencing method was used for comparison. RESULTS: With spiking synthetic mutant DNA in wildtype GM24385 cell line DNA, this assay can detect UBA1 mutations with a detection limit of variant allele frequency (VAF) at 0.1-5%. Our assay shows 100% concordance with Sanger sequencing results when used for analyzing 15 positive and 19 negative clinical samples. CONCLUSIONS: The QClamps® Plex UBA1 Mutation Detection Assay is a quicker, simpler, and more sensitive assay that can accurately detect the UBA1 mutations even at early stages with low mutation frequency.

Testing and vaccination have been major components of the strategy for combating the ongoing COVID-19 pandemic. In this study, we have developed a quantitative anti-SARS-CoV-2 spike (S1) IgG antibody assay using a fingerstick dried blood sample. We evaluated the feasibility of using this high-throughput and quantitative anti-SARS-CoV-2 spike (S1) IgG antibody testing assay in vaccinated individuals. Fingerstick blood samples were collected and analyzed from 137 volunteers before and after receiving the Moderna or Pfizer mRNA vaccine. Anti-SARS-CoV-2 S1 IgG antibody could not be detected within the first 7 days after receiving the first vaccine dose, however, the assay reliably detected antibodies from day 14 onwards. In addition, no anti-SARS-CoV-2 nucleocapsid (N) protein IgG antibody was detected in any of the vaccinated or healthy participants, indicating that the anti-SARS-CoV-2 S1 IgG assay is specific for the mRNA vaccine-induced antibodies. The S1 IgG levels detected in fingerstick samples correlated with the levels found in venous blood plasma samples and with the efficacy of venous blood plasma samples in the plaque reduction neutralization test (PRNT). The assay displayed a limit of quantification (LOQ) of 0.59 μg/mL and was found to be linear in the range of 0.51-1000 μg/mL. Finally, its clinical performance displayed a Positive Percent Agreement (PPA) of 100% (95% CI: 0.89-1.00) and a Negative Percent Agreement (NPA) of 100% (95% CI: 0.93-1.00). In summary, the assay described here represents a sensitive, precise, accurate, and simple method for the quantitative detection and monitoring of post-vaccination anti-SARS-CoV-2 spike IgG responses.

BACKGROUND: Serology tests for detecting the antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can identify previous infection and help to confirm the presence of current infection. OBJECTIVE: The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgG antibody detection. RESULTS: Clinical agreement studies were performed in 107 COVID-19 patient serum samples and 226 negative donor serum/plasma samples. Positive percent agreement (PPA) was 46.15 % (95 % CI: 19.22 % ∼74.87 %), 61.54 % (95 % CI: 31.58 % ∼86.14 %), and 97.53 % (95 % CI: 91.36 % ∼99.70 %) for samples collected on 0-7 days, 8-14 days, and ≥15 days from symptom onset, respectively. Negative Percent Agreement (NPA) was 98.23 % (95 % CI: 95.53 % ∼99.52 %). No cross-reactivity was observed to patient samples positive for IgG antibodies against the following pathogens: HIV, HAV, HBV, RSV, CMV, EBV, Rubella, Influenza A, and Influenza B. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. CONCLUSION: An anti-SARS-CoV-2 IgG antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up anti-SARS-CoV-2 IgG testing.

BACKGROUND: Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling. METHODS: Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated. RESULTS: The LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100-200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%). CONCLUSION: The studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.

Introduction: Colorectal cancer screening programs are effective in reducing incidence and mortality. In Europe, every FIT+ patient is referred to colonoscopy. The available data show that ~75.0% of these patients are negative. It is desirable to select patients at a greater risk of having a positive colonoscopy. Materials and Methods: 711 subjects, aged 50–74, attending the screening program of ASL-NA-3-SUD (Naples, Italy), were enrolled in a cross-sectional study to evaluate the performance of the QuantiDNA™ test and the non-inferiority of an alternative approach (AAP). This evaluation is based on FIT+ and QuantiDNA™+ patients referred to colonoscopy, compared to Standard of Care (SOC) colonoscopy following a FIT+ test alone. A non-inferiority margin (NIM) for colorectal neoplasia (CN) and advanced adenomas (AA) was set at −10% and at −3.8% for CRC. Results: The odds ratio was 1.76 (p-value = 0.009). The detection rate of AAP was 15.9% for colorectal neoplasia, 13.0% for advanced adenoma, and 3.0% for CRC. The risk difference between AAP and SOC was −5.07% (95% C.I. −9.23, −0.90) for colorectal neoplasia, −4.02% (95% C.I. −7.89, −0.16) for advanced adenomas, and −1.04% (95% C.I. −3.16, 1.07) for CRC. This data suggests that AAP is non-inferior to SOC for detecting CN, AA, and CRC. The expected decrease in colonoscopies is 33.4%. Conclusions: The QuantiDNA™ test is straightforward, non-invasive, and well-tolerated. Data from this study indicate that it is effective in the reduction of the need for colonoscopy examinations (−33.4%) and is non-inferior to SOC in the detection of significant colorectal lesions. [ABSTRACT FROM AUTHOR]

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5–7%, 1–2%, and 1–2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability. [ABSTRACT FROM AUTHOR]

Ultrasonic methods facilitate the determination of the elastic constants of fluorapatite. The elastic constant reveals that fluorapatite is highly anisotropic in structure. The elastic properties are dominated by hierarchical bicomposite microstructure of the human bone, which is made of fluorapatite.

A novel application of Nanoplex biotags is described for the direct detection of rare cancer cells in whole blood. Multiplexed labeling of biomarkers with multiplex surface-enhanced Raman scattering (SERS) tags has improved the accuracy and specificity of assay.

Various probe designs and experimental parameters for no-wash, sealed chamber and multiplexed detection of nucleic acids by combining molecular beacon (MB) probes, as well as the barcoded metal nanowires are discussed. The results show that such nanowires based MB assays are extremely helpful, as they help in reducing assay complexity and the risk of contamination to a very large extent and hence require no further sample manipulations.