Solid-phase PCR based on thermostable, encoded magnetic microspheres for simple, highly sensitive and multiplexed nucleic acid detection

Objective To explore the usefulness of highly sensitive nucleic acid detection for assisting with the accurate antiviral treatment of patients with cirrhosis that was caused by hepatitis. Methods There were 377 patients with hepatitis B with cirrhosis and 119 patients with hepatitis C with cirrhosis, either as hospitalized patients and outpatients, who were enrolled into the study. Among them, 299 were men and 197 were women between 23 and 82 years of age. All patients were examined using a domestic HBV DNA/HCV RNA test, which was negative in 162 cirrhosis with hepatitis B and 54 cirrhosis with hepatitis C patients (HBV DNA/HCV RNA <500 IU/mL). Prediction and analysis of the HBV DNA load using alanine aminotransferase (ALT) level was based on receiver operating characteristics (ROC) curve analysis. Results For patients with hepatitis C with cirrhosis, after the antiviral therapy, ALT, HCV RNA, and Child–Pugh grade were significantly improved compared with before treatment. ROC analysis results showed that an ALT level of 29 IU/mL was the most sensitive cutoff value to judge a positive HBV DNA load (sensitivity 1.0, specificity 0.237, Youden index 0.763). Conclusion Precise detection for patients with cirrhosis caused by hepatitis is required for accurate therapy.

Download Full-text

CRISPR-based surveillance for COVID-19 using genomically-comprehensive machine learning design

10.1101/2020.02.26.967026 ◽

2020 ◽

Cited By ~ 41

Author(s):

Hayden C. Metsky ◽

Catherine A. Freije ◽

Tinna-Solveig F. Kosoko-Thoroddsen ◽

Pardis C. Sabeti ◽

Cameron Myhrvold

Keyword(s):

Nucleic Acid ◽

Clinical Presentation ◽

Detection System ◽

Genomic Diversity ◽

Nucleic Acid Detection ◽

Learning Design ◽

Highly Sensitive ◽

Global Concern ◽

Ongoing Surveillance ◽

Flow Detection

AbstractThe emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly become a global concern and has highlighted the need for fast, sensitive, and specific tools to surveil circulating viruses. Here we provide assay designs and experimental resources, for use with CRISPR-based nucleic acid detection, that could be valuable for ongoing surveillance. We provide assay designs for detection of 67 viral species and subspecies, including: SARS-CoV-2, phylogenetically-related viruses, and viruses with similar clinical presentation. The designs are outputs of algorithms that we are developing for rapidly designing nucleic acid detection assays that are comprehensive across genomic diversity and predicted to be highly sensitive and specific. Of our design set, we experimentally screened 4 SARS-CoV-2 designs with a CRISPR-Cas13 detection system and then extensively tested the highest-performing SARS-CoV-2 assay. We demonstrate the sensitivity and speed of this assay using synthetic targets with fluorescent and lateral flow detection. Moreover, our provided protocol can be extended for testing the other 66 provided designs. Assay designs are available at https://adapt.sabetilab.org/.

Download Full-text

Quantitative polymerase chain reaction and solid-phase capture nucleic acid detection

Bioluminescence and Chemiluminescence Part C - Methods in Enzymology ◽

10.1016/s0076-6879(00)05507-5 ◽

2000 ◽

pp. 466-476

Author(s):

Chris S. Martin ◽

John C. Voyta ◽

Irena Bronstein

Keyword(s):

Polymerase Chain Reaction ◽

Nucleic Acid ◽

Solid Phase ◽

Quantitative Polymerase Chain Reaction ◽

Nucleic Acid Detection ◽

Chain Reaction ◽

Polymerase Chain

Download Full-text

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid and Potential Utility for SARS-CoV-2 Detection

10.1101/2021.08.30.458287 ◽

2021 ◽

Author(s):

Xiaoyu Zhang ◽

Venubabu Kotikam ◽

Eriks Rozners ◽

Brian P. Callahan

Keyword(s):

Nucleic Acid ◽

Solid Phase ◽

Detection Sensitivity ◽

High Yield ◽

Nucleic Acid Detection ◽

Phase Synthesis ◽

Dna Oligonucleotides ◽

C Terminus ◽

Potential Use ◽

Better Than

Enzymatic beacons, or E-beacons, are 1:1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming DNA oligonucleotides equipped with a dark quencher. We prepared E-beacons biocatalytically using the promiscuous hedgehog protein-cholesterol ligase, HhC. Instead of cholesterol, HhC attached nanoluciferase site-specifically to mono-sterylated hairpin DNA, prepared in high yield by solid phase synthesis. We tested three potential E-beacon dark quenchers: Iowa Black, Onyx-A, and dabcyl. Prototype E-beacon carrying each of those quenchers provided sequence-specific nucleic acid sensing through turn-on bioluminescence. For practical application, we prepared dabcyl-quenched E-beacons for potential use in detecting the COVID-19 virus, SARS-CoV-2. Targeting the E484 codon of the SARS-CoV-2 Spike protein, E-beacons (80 x 10-12 M) reported wild-type SARS-CoV-2 nucleic acid at greater than or equal to 1 x 10-9 M with increased bioluminescence of 8-fold. E-beacon prepared for the E484K variant of SARS-CoV-2 functioned with similar sensitivity. These E-beacons could discriminate their complementary target from nucleic acid encoding the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with specificity, detection sensitivity with E-beacons is two to three orders of magnitude better than synthetic molecular beacons, rivaling the most sensitive nucleic acid detection agents re-ported to date.

Download Full-text