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

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.

scholarly journals DNA recovery from Droplet Digital™ PCR emulsions using liquid nitrogen

BioTechniques ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 450-454
Author(s):  
Lara Dutra ◽  
Ole Franz ◽  
Veli-Mikko Puupponen ◽  
Marja Tiirola
Keyword(s):  
Phase Separation ◽  
Liquid Nitrogen ◽  
Sensitivity And Specificity ◽  
Dna Content ◽  
High Sensitivity ◽  
Droplet Microfluidics ◽  
Rapid Freezing ◽  
Dna Recovery ◽  
Pcr Products ◽  
Pcr Assays

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.

scholarly journals Variola Virus-Specific Diagnostic Assays: Characterization, Sensitivity, and Specificity

2015 ◽  
Vol 53 (4) ◽  
pp. 1406-1410 ◽  
Author(s):  
Ashley V. Kondas ◽  
Victoria A. Olson ◽  
Yu Li ◽  
Jason Abel ◽  
Miriam Laker ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Variola Virus ◽  
Public Health Response ◽  
Diagnostic Assays ◽  
Health Response ◽  
Pcr Assays

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.

scholarly journals RelocaTE2: a high resolution transposable element polymorphism mapping tool for population resequencing

2016 ◽  
Author(s):  
Jinfeng Chen ◽  
Travis Wrightsman ◽  
Susan R Wessler ◽  
Jason E. Stajich
Keyword(s):  
Transposable Element ◽  
Sensitivity And Specificity ◽  
Genome Sequencing ◽  
Population Genetic ◽  
Sequence Data ◽  
False Positive Rate ◽  
Rice Chromosome ◽  
High Sensitivity ◽  
Real Sequence ◽  
Robust Solution

Background Transposable element (TE) polymorphisms are important components of population genetic variation. The functional impacts of TEs in gene regulation and generating genetic diversity have been observed in multiple species, but the frequency and magnitude of TE variation is under appreciated. Inexpensive and deep sequencing technology has made it affordable to apply population genetic methods to whole genomes with methods that identify single nucleotide and insertion/deletion polymorphisms. However, identifying TE transposition events or polymorphisms can be challenging due to the repetitive nature of these sequences, which hamper both the sensitivity and specificity of analysis tools. Methods We have developed the tool RelocaTE2 ( http://github.com/stajichlab/RelocaTE2 ) for identification of TE polymorphisms at high sensitivity and specificity. RelocaTE2 searches for known TE sequences in whole genome sequencing reads from second generation sequencing platforms such as Illumina. These sequence reads are used as seeds to pinpoint chromosome locations where TEs have transposed. RelocaTE2 detects target site duplication (TSD) of TE insertions allowing it to report TE polymorphism loci with single base pair precision. Results and Discussion The performance of RelocaTE2 is evaluated using both simulated and real sequence data. RelocaTE2 demonstrates a higher level of sensitivity and specificity when compared to other tools. Even in highly repetitive regions, such as those tested on rice chromosome 4, RelocaTE2 is able to report up to 95% of simulated TE insertions with less than 0.1% false positive rate using 10-fold genome coverage resequencing data. RelocaTE2 provides a robust solution to identify TE polymorphisms and can be incorporated into analysis workflows in support of describing the complete genotype from light coverage genome sequencing.

scholarly journals CADM1, MAL, and miR124 Promoter Methylation as Biomarkers of Transforming Cervical Intrapithelial Lesions

2019 ◽  
Vol 20 (9) ◽  
pp. 2262 ◽  
Author(s):  
Marta del Pino ◽  
Adriana Sierra ◽  
Lorena Marimon ◽  
Cristina Martí Delgado ◽  
Adriano Rodriguez-Trujillo ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Promoter Methylation ◽  
Methylation Status ◽  
High Sensitivity ◽  
Control Group ◽  
Low Grade ◽  
Papilloma Virus ◽  
Specific Pcr ◽  
Risk Of Progression ◽  
Intraepithelial Lesions

Background: Squamous intraepithelial lesions/cervical intraepithelial neoplasias (SIL/CIN) are high-risk human papilloma virus (hrHPV)-related lesions which are considered as high grade (HSIL/CIN2-3) or low grade (LSIL/CIN1) lesions according to their risk of progression to cervical cancer (CC). Most HSIL/CIN2-3 are considered as transforming hrHPV infections, so truly CC precursors, although some clear spontaneously. hrHPV testing has a high sensitivity for the detection of HSIL/CIN2-3 but a relatively low specificity for identifying transforming lesions. We aimed to determine whether the combination of CADM1, MAL and miR124 promoter methylation status assessed in histological samples can be used as a biomarker in the identification of transforming HSIL/CIN lesions. Design: 131 cervical biopsies, including 8 cases with no lesion and a negative hrHPV test result (control group), 19 low-grade (L)SIL/CIN1, 30 HSIL/CIN2, 60 HSIL/CIN3, and 14 CC were prospectively collected. hrHPV was detected and genotyped using the polymerase chain reaction (PCR)-based technique SPF10 HPV LIPA. A multiplex quantitative methylation-specific PCR (qMSP) was used to identify the methylation status of the CADM1, MAL, and miR124 promoter genes. Results: Significantly higher methylation levels of CADM1, MAL and miR-124 were found in HSIL/CIN2-3 and CC compared with normal and LSIL lesions. DNA methylation of at least one gene was detected in 12.5% (1/8) of normal samples, 31.5% (6/19) of LSIL/CIN1, 83.3% (25/30) of HSIL/CIN2, 81.6% (49/60) of HSIL/CIN3 and 100% (14/14) of CC (p < 0.001). The sensitivity and specificity for HSIL/CIN2-3 and CC of having at least one methylated gene were 84.6% and 74.0%, respectively. The sensitivity and specificity of the combination of at least one methylated gene and a positive hrHPV test were 80.7% and 85.1% for HSIL/CIN2-3 and CC, respectively. Conclusions: The methylation rate of CADM1, MAL and miR124 increases with the severity of the lesion. Further research is warranted to evaluate the usefulness of these biomarkers for the identification of transforming HSIL/CIN.

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

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

Trends in Diagnosis for Active Tuberculosis Using Nanomaterials

2019 ◽  
Vol 26 (11) ◽  
pp. 1946-1959 ◽  
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.

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

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.

scholarly journals Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Ellen N. Kersh ◽  
Cau D. Pham ◽  
John R. Papp ◽  
Robert Myers ◽  
Richard Steece ◽  
...  
Keyword(s):  
Public Health ◽  
Antibiotic Resistance ◽  
Neisseria Gonorrhoeae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Whole Genome ◽  
Content Type ◽  
Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

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

Diagnostics ◽  
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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