DNA recovery from Droplet Digital™ PCR emulsions using liquid nitrogen

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.

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Regional and temporal variations affect the accuracy of variant-specific SARS-CoV-2 PCR assays

10.1101/2021.11.08.21266083 ◽

2021 ◽

Author(s):

Chamteut Oh ◽

Palash Sashittal ◽

Aijia Zhou ◽

Leyi Wang ◽

Mohammed El-Kebir ◽

...

Keyword(s):

Public Health ◽

Sensitivity And Specificity ◽

Genome Sequencing ◽

High Sensitivity ◽

Temporal Variations ◽

Health Decisions ◽

Specific Pcr ◽

Geographical Regions ◽

Design Variant ◽

Pcr Assays

Monitoring the prevalence of SARS-CoV-2 variants is necessary to make informed public health decisions during the COVID-19 pandemic. PCR assays have received global attention, facilitating rapid understanding of variant dynamics because they are more accessible and scalable than genome sequencing. However, as PCR assays target only a few mutations, their accuracy could be compromised when these mutations are not exclusive to target variants. Here we show how to design variant-specific PCR assays with high sensitivity and specificity across different geographical regions by incorporating sequences deposited in the GISAID database. Furthermore, we demonstrate that several previously developed PCR assays have decreased accuracy outside their study areas. We introduce PRIMES, an algorithm that enables the design of reliable PCR assays, as demonstrated in our experiments that enabled tracking of dominant SARS-CoV-2 variants in local sewage samples. Our findings will contribute to improving PCR assays for SARS-CoV-2 variant surveillance.

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18S Ribosomal DNA-Based PCR for Diagnosis ofTrichomonas vaginalis

Journal of Clinical Microbiology ◽

10.1128/jcm.38.7.2683-2687.2000 ◽

2000 ◽

Vol 38 (7) ◽

pp. 2683-2687 ◽

Cited By ~ 69

Author(s):

Holger Mayta ◽

Robert H. Gilman ◽

Maritza M. Calderon ◽

Aren Gottlieb ◽

Giselle Soto ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Trichomonas Vaginalis ◽

High Sensitivity ◽

18S Rrna Gene ◽

Vaginal Swab ◽

Urine Samples ◽

Rrna Gene ◽

Sexually Transmitted ◽

Immunodeficiency Virus ◽

Pcr Products

Trichomonas vaginalis remains the most common sexually transmitted parasite in the world and is considered a major risk factor in the transmission of the human immunodeficiency virus. A PCR technique using primers targeting a specific region of the 18S rRNA gene of T. vaginalis was developed. The PCR test was standardized using 15 reference strains, giving a single product of 312 bp in all strains. No amplification was observed when DNA from related organisms or human DNA was used as a target. The test was evaluated on 372 vaginal swab specimens and 361 urine samples from women attending infertility and obstetric clinics at two separate hospitals in Lima, Peru. Compared to T. vaginalis culture, the overall sensitivity and specificity of PCR of vaginal swab samples was 100% and 98%, respectively. The PCR of urine samples was 100% sensitive and 99.7% specific compared to culture of vaginal swab, but the sensitivity drops to 83.3% when compared to PCR of vaginal swabs. All culture-positive samples were found to be positive by PCR in either urine or vaginal secretion. None of the PCR-negative samples were positive by culture. The origin of the amplification was confirmed by digestion of PCR products with HaeIII. This PCR assay, which is easy to perform and has a high sensitivity and specificity, should be useful for routine diagnosis of T. vaginalisinfection.

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Detector-C: A Blood-based IVD with High Sensitivity and Specificity for Early Detection and Screening of Colorectal Cancer

Zeitschrift für Gastroenterologie ◽

10.1055/s-0030-1263628 ◽

2010 ◽

Vol 48 (08) ◽

Author(s):

A Rosenthal ◽

H Köppen ◽

R Musikowski ◽

R Schwanitz ◽

J Behrendt ◽

...

Keyword(s):

Colorectal Cancer ◽

Early Detection ◽

Sensitivity And Specificity ◽

High Sensitivity

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Trends in Diagnosis for Active Tuberculosis Using Nanomaterials

Current Medicinal Chemistry ◽

10.2174/0929867325666180912105617 ◽

2019 ◽

Vol 26 (11) ◽

pp. 1946-1959 ◽

Cited By ~ 3

Author(s):

Le Minh Tu Phan ◽

Lemma Teshome Tufa ◽

Hwa-Jung Kim ◽

Jaebeom Lee ◽

Tae Jung Park

Keyword(s):

Sensitivity And Specificity ◽

High Efficiency ◽

Point Of Care ◽

High Sensitivity ◽

Signs And Symptoms ◽

Diagnostic Methods ◽

Advantages And Disadvantages ◽

Treatment And Prevention ◽

Clinical Tools ◽

Diagnostic Applications

Background:Tuberculosis (TB), one of the leading causes of death worldwide, is difficult to diagnose based only on signs and symptoms. Methods for TB detection are continuously being researched to design novel effective clinical tools for the diagnosis of TB.Objective:This article reviews the methods to diagnose TB at the latent and active stages and to recognize prospective TB diagnostic methods based on nanomaterials.Methods:The current methods for TB diagnosis were reviewed by evaluating their advantages and disadvantages. Furthermore, the trends in TB detection using nanomaterials were discussed regarding their performance capacity for clinical diagnostic applications.Results:Current methods such as microscopy, culture, and tuberculin skin test are still being employed to diagnose TB, however, a highly sensitive point of care tool without false results is still needed. The utilization of nanomaterials to detect the specific TB biomarkers with high sensitivity and specificity can provide a possible strategy to rapidly diagnose TB. Although it is challenging for nanodiagnostic platforms to be assessed in clinical trials, active TB diagnosis using nanomaterials is highly expected to achieve clinical significance for regular application. In addition, aspects and future directions in developing the high-efficiency tools to diagnose active TB using advanced nanomaterials are expounded.Conclusion:This review suggests that nanomaterials have high potential as rapid, costeffective tools to enhance the diagnostic sensitivity and specificity for the accurate diagnosis, treatment, and prevention of TB. Hence, portable nanobiosensors can be alternative effective tests to be exploited globally after clinical trial execution.

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Value of Coronary Calcium Scoring in Symptomatic and Asymptomatic Coronary Artery Disease Patients

Current Medical Imaging Formerly Current Medical Imaging Reviews ◽

10.2174/1573405616666201023142030 ◽

2020 ◽

Vol 16 ◽

Author(s):

Hala T. Salem ◽

Eman A.S. Sabek

Keyword(s):

Coronary Artery Disease ◽

Coronary Artery ◽

Sensitivity And Specificity ◽

High Sensitivity ◽

Coronary Calcium ◽

Risk Estimate ◽

Calcium Scoring ◽

Coronary Calcium Scoring ◽

Framingham Risk ◽

Artery Disease

Aim and Objective: To estimate the relationship between Coronary Calcium Scoring (CCS)and presence of different degrees of obstructive coronary artery disease (CAD) to avoid unnecessary examinations and hence unnecessary radiation exposure and contrast injection. Background: Coronary Calcium Scoring (CCS) is a test uses x-ray equipment to produce pictures of the coronary arteries to determine the degree of its narrowing by the build-up of calcified plaques. Despite the lack of definitive data linking ionizing radiation with cancer, the American Heart Association supports widely that practitioners of Computed tomography Coronary Angiography (CTCA) should keep “patient radiation doses as low as reasonably achievable but consistent with obtaining the desired medical information”. Methods: Data obtained from 275 CTCA examinations were reviewed. Radiation effective doses were estimated for both CCS and CTCA, measures to keep it as low as possible were presented, CCS and Framingham risk estimate were compared to the final results of CTCA to detect sensitivity and specificity of each one in detecting obstructive lesions. Results: CCS is a strong discriminator for obstructive CAD and can with high sensitivity and specificity and correlates well with the degree of obstruction even more than Framingham risk estimate which has high sensitivity and low specificity. Conclusion: CCS helps reducing the effective radiation dose if properly evaluated to skip unnecessary CTCA if obstructive lesions was unlikely, and as a test does not use contrast material, harmful effect on the kidney will be avoided as most of coronary atherosclerotic patients have renal problems.

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Blood-Based Detection of Colorectal Cancer Using Cancer-Specific DNA Methylation Markers

Diagnostics ◽

10.3390/diagnostics11010051 ◽

2020 ◽

Vol 11 (1) ◽

pp. 51

Author(s):

Nam-Yun Cho ◽

Ji-Won Park ◽

Xianyu Wen ◽

Yun-Joo Shin ◽

Jun-Kyu Kang ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Sensitivity And Specificity ◽

Stage Iv ◽

High Sensitivity ◽

Liquid Biopsies ◽

Blood Leukocytes ◽

Marker Panel ◽

A Genome ◽

Methylight Assay

Cancer tissues have characteristic DNA methylation profiles compared with their corresponding normal tissues that can be utilized for cancer diagnosis with liquid biopsy. Using a genome-scale DNA methylation approach, we sought to identify a panel of DNA methylation markers specific for cell-free DNA (cfDNA) from patients with colorectal cancer (CRC). By comparing DNA methylomes between CRC and normal mucosal tissues or blood leukocytes, we identified eight cancer-specific methylated loci (ADGRB1, ANKRD13, FAM123A, GLI3, PCDHG, PPP1R16B, SLIT3, and TMEM90B) and developed a five-marker panel (FAM123A, GLI3, PPP1R16B, SLIT3, and TMEM90B) that detected CRC in liquid biopsies with a high sensitivity and specificity with a droplet digital MethyLight assay. In a set of cfDNA samples from CRC patients (n = 117) and healthy volunteers (n = 60), a panel of five markers on the platform of the droplet digital MethyLight assay detected stages I–III and stage IV CRCs with sensitivities of 45.9% and 95.7%, respectively, and a specificity of 95.0%. The number of detected markers was correlated with the cancer stage, perineural invasion, lymphatic emboli, and venous invasion. Our five-marker panel with the droplet digital MethyLight assay showed a high sensitivity and specificity for the detection of CRC with cfDNA samples from patients with metastatic CRC.

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