A universal linker-RT PCR based quantitative method for the detection of circulating miRNAs

2014 ◽  
Vol 6 (22) ◽  
pp. 9101-9107 ◽  
Author(s):  
Qinyu Ge ◽  
Fei Tian ◽  
Youxia Zhou ◽  
Yanan Zhu ◽  
Jiafeng Lu ◽  
...  
Keyword(s):  
Quantitative Method ◽  
Circulating Mirnas ◽  
Rt Pcr ◽  
Qpcr Method ◽  
Higher Sensitivity

A higher sensitivity and reliable qPCR method based on a random pre-adenylated DNA adaptor ligation was developed and validated for the detection of circulating miRNAs.

scholarly journals Comparative evaluation of cost effective extraction free molecular technique for detection of SARS-CoV-2 with reference to standard VTM based RT-qPCR method

2021 ◽  
Author(s):  
Megh Singh Dhakad ◽  
Sanjib Gogoi ◽  
Ansu Kumari ◽  
Aashish Kumar Singh ◽  
Manoj B. Jais ◽  
...  
Keyword(s):  
Detection Method ◽  
Cost Effective ◽  
Molecular Technique ◽  
Rt Pcr ◽  
Index Method ◽  
Resource Limited ◽  
Qpcr Method ◽  
Kappa Value ◽  
The Cost ◽  
Higher Sensitivity

Background and Objectives: The entire globe is undergoing an unprecedented challenge of COVID-19. Considering the need of rapid and accurate diagnostic tests for SARS-CoV-2, this study was planned to evaluate the cost effective extraction free RT-PCR technique in comparison to the standard VTM based RT-qPCR method. Materials and Methods: Paired swabs from nasopharynx and oropharynx were collected for SARS-CoV-2 testing, from 211 adult patients (≥18 years) in VTM and plain sterile tubes (dry swabs). These samples were processed and RT-qPCR was carried out as per standard protocols. Results: 54.5% of the patients were females and 45.5% were males with sex ratio 1:1.19 (M: F). 38.86% were symptomatic, of which fever (86.59%), cough (79.23%) and breathlessness (46.34%) were the most common symptoms. The positivity by VTM based method and index method was 31.27% and 13.27% respectively. Of the 27 inconclusive results from index method, 37.04% were positive, 48.15% were negative by VTM based method. However, in 40 inconclusive results by VTM based method, 90% were negative and rest remained inconclusive by index method. The sensitivity and specificity of the index method were 39.39% and 85.71% respectively. The overall agreement between VTM based method and index method was 49.59% with estimated Kappa value of 0.19. Conclusion: VTM based method showed higher sensitivity compared to the index method. The higher positivity by VTM based method, suggests that VTM based method could plausibly be a better detection method of SARS-CoV-2. Still, the index method might add value in a resource limited setups for detection of SARS-CoV-2.  

scholarly journals Rapid, Reliable and Robust approach for extraction-free RT-PCR based detection of SARS-CoV-2 in clinical setting to expedite large scale screening

2021 ◽  
Author(s):  
Abhilasha Dubey ◽  
Sanjay Upadhyay ◽  
Manjeet Mehta
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
Low Cost ◽  
Diagnostic Methods ◽  
Analysis Method ◽  
Rt Pcr ◽  
Qpcr Method ◽  
Quantitative Results ◽  
Pooled Samples ◽  
Large Scale Screening

Rapid, reliable and robust method for the detection of SARS-CoV-2 is an indispensable need for diagnostics. The development of diagnostic methods will aid to address further waves of the pandemic potentially with rapid surveillance of disease and to allay the fears. To meet this challenge, we have developed a rapid RT-qPCR method for the detection of 3 target genes or confirmatory genes in less than 30 minutes. The assay showed 100% sensitivity and 100% specificity when tested on 120 samples. We compared a conventional extraction based method with extraction-free method, and then further reduced the run time of extraction free method. Additionally, we have validated our rapid RT-qPCR method for the assessment of pooled samples. We hereby propose a most reliable approach for the mass screening of samples with ease of operation at a low cost. Finally we designed a single tube analysis method which provides qualitative as well as quantitative results in minimum time.

scholarly journals COMPARISON OF ARTIFICIAL INTELLIGENCE ENABLED METHODS IN THE COMPUTED TOMOGRAPHIC ASSESSMENT OF COVID-19 DISEASE.

2020 ◽  
Author(s):  
Atul Kapoor ◽  
Goldaa Mahajan ◽  
Aprajita Kapoor
Keyword(s):  
Artificial Intelligence ◽  
Sensitivity And Specificity ◽  
Visual Analysis ◽  
Test Results ◽  
Friedman Test ◽  
Rt Pcr ◽  
Computed Tomographic ◽  
Sensitivity Specificity ◽  
Disease Parameters ◽  
Higher Sensitivity

Objectives: Comparison of three different Artificial intelligence (AI) methods of assessment for patients undergoing Computed tomography (CT) for suspected Covid-19 disease. Parameters studied were probability of diagnosis, quantification of disease severity and the time to reach the diagnosis . Methods: 107 consecutive patients of suspected Covid-19 patients were evaluated using the three AI methods labeled as AI-I,II, III alongwith visual analysis labeled as VT for predicting probability of Covid-19, determining CT severity score (CTSS) and index (CTSI) , percentage opacification (PO) and high opacification (POHO). Sensitivity, specificity along with area under curves were estimated for each method and the CTSS and CTSI correlated using Friedman test. Results: Out of 107 patients 71 patients were Covid-19 positive and 20 negative by RT-PCR while 16 did not get RT-PCR done. AI-III method showed higher sensitivity and specificity of 93% and 88% respectively to predict probability of Covid 19. It had 2 false positive patients of interstitial lung disease. AI-II method had sensitivity and specificity of 66% and 83% respectively while visual (VT) analysis showed sensitivity and specificity of 59.7% and 62% respectively. Statistically significant differences were also seen in CTSI and PO estimation between AI-I and III methods (p<0.0001) with AI-III showing fastest time to calculate results. Conclusions: AI-III method gave better results to make an accurate and quick diagnosis of the Covid-19 with AUC of 0.85 to predict probability of Covid-19 alongwith quantification of Covid-19 lesions in the form of PO, POHO as compared to other AI methods and also by visual analysis.

scholarly journals COVID- 19 Screening by RT-PCR: An Epidemiological Modelling

2020 ◽  
Author(s):  
Ali A. Hasab
Keyword(s):  
Clinical Symptoms ◽  
Case Fatality ◽  
Secondary Data ◽  
Reproductive Rate ◽  
Rt Pcr ◽  
Roc Curve Analysis ◽  
Epidemic Dynamics ◽  
Sir Epidemic ◽  
The Impact ◽  
Higher Sensitivity

Abstract Background: Given the attention on COVID-19 testing and its role in helping to halt the spread of COVID-19 Pandemic, wider testing is urgently needed for successful pandemic control. The level of the test’s performance is also important for effective management of the different stages of the pandemic.Objectives: To study the impact of RT-PCR testing in control of COVID-19 Pandemic and validity of RT-PCR as a predictor for COVID- 19 diseaseMethods: The data was collected essentially by using secondary data. All cases and deaths in WHO Situation Reports and total tests in Worldometer were included in the study. Wolfram Player 12 software was used for the Susceptible Infected Recovered (SIR) epidemic dynamics of COVID-19. Survival analysis was carried out to determine the cumulative proportional survival of COVID-19 in Egypt. Six studies discussing the validity of RT-PCR was also reanalyzed. Receiver Operating Characteristic (ROC) curve analysis was used to study the diagnostic performance of RT-PCR.Results: There was a negative correlation between both case fatality rate of COVID-19 and reproductive rate with RT-PCR tests performed . This difference is significant (r = - 0.307 and. – 0.361) respectively. RT-PCR had a sensitivity of 61.19%, Specificity 94.75%% and an accuracy of 76.72%. The area under the ROC (AUC) for RT-PCR was 0.780.Conclusion and recommendation: RT-PCR testing will continue to be needed. It reduced the case fatality and reproduction rates of COVID-19 Pandemic. The AUC for RT-PCR is less than optimal. The combination of clinical symptoms, exposure history and CT must be considered to identify COVID-19 with higher sensitivity.

Comparison of different primer sets for the RT-PCR detection of hepatitis A virus and astrovirus in mussel tissues

2004 ◽  
Vol 50 (1) ◽  
pp. 131-136 ◽  
Author(s):  
J.L. Romalde ◽  
C. Ribao ◽  
M. Luz Vilariño ◽  
J.L. Barja
Keyword(s):  
Detection Limit ◽  
Critical Point ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Primer Sets ◽  
Selection Of ◽  
Higher Sensitivity

In the present study, the efficiency of several primer sets for the RT-PCR detection of hepatitis A virus (HAV) and astrovirus from both crude viral extracts and experimentally infected shellfish tissues was evaluated. Differences were observed depending on the primer set employed in the sensitivity of amplification of both viral types. For HAV primers, HAV240/HAV68 yielded the higher sensitivity: showing a detection limit of 0.02-0.1 infectious particles/μL or mg of tissue (either crude extracts or seeded mussel tissues). Regarding detection of AsV, a better performance was observed with primer set A1/A2 achieving a sensitivity of 0.1-1 PFU/μL or mg of tissue. The results obtained in this work strongly indicated that selection of primer sets to be employed for the routine detection of enteric viruses was a critical point in the design of the RT-PCR protocols.

scholarly journals Predictive Value of Circulating miRNAs in Lymph Node Metastasis for Colon Cancer

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 176
Author(s):  
In Hee Lee ◽  
Gyeonghwa Kim ◽  
Sang Gyu Kwak ◽  
Dong Won Baek ◽  
Byung Woog Kang ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Lymph Node ◽  
Cancer Patients ◽  
Circulating Mirnas ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Diagnosis And Prognosis ◽  
Polymerase Chain ◽  
Validation Set ◽  
Plasma Mirna

(1) Background: Lymph node (LN) status is an indubitable prognostic factor for survival among colon cancer patients. MicroRNAs (miRNAs) have been implicated in the development and progression of many cancers and are potential biomarkers for cancer diagnosis and prognosis. Therefore, we validated candidate biomarkers using circulating miRNAs by analyzing the plasma miRNA concentrations from patients with colon cancer to predict LN metastasis. (2) Methods: This study included 79 blood samples from patients diagnosed with colon cancer. The NanoString assay was used for screening, and TaqMan miRNA assays for quantitative real-time polymerase chain reaction (RT-PCR) test was used for validation. In a discovery set, we compared the expression of 800 circulating miRNAs in 24 samples (stage 0/I/IIA versus IIIB/IIIC). For validation, a total 79 samples were tested using quantitative RT-PCR. (3) Results: In the discovery set, 10 candidate circulating miRNAs were detected (4 up-regulated miRNAs: miR-323a-3p, miR-382-5p, miR-29a-3p, and miR-376a-3p; 6 down-regulated miRNAs: miR-26a-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-374a-5p, and let-7b-5p). In the validation set, higher expression of three circulating miRNAs (miR-323a-3p, miR-382-5p, and miR-376a-3p) was significantly associated with LN metastasis (p = 0.0063, 0.0107, and 0.0022). (4) Conclusions: High expression of circulating miR-323a-3p, miR-382-5p, and miR-376a-3p was significantly associated with LN metastasis in colon cancer patients. These miRNAs could be circulating biomarker candidates that predict the presence of LN metastasis.

scholarly journals Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs

2020 ◽  
Vol 31 (1) ◽  
Author(s):  
Ankica Sekovanić ◽  
Adrijana Dorotić ◽  
Jasna Jurasović ◽  
Daria Pašalić ◽  
Jelena Kovačić ◽  
...  
Keyword(s):  
Pcr Analysis ◽  
Circulating Mirnas ◽  
Rt Pcr

scholarly journals High prevalence of GB Virus C/Hepatitis G Virus RNA among Brazilian blood donors

2003 ◽  
Vol 45 (2) ◽  
pp. 75-78 ◽  
Author(s):  
José Eduardo Levi ◽  
Daniela Gazoto Contri ◽  
Liliam Pereira Lima ◽  
Deise Tihe Takaoka ◽  
Regina Helena Garrini ◽  
...  
Keyword(s):  
Blood Donors ◽  
Place Of Birth ◽  
Rt Pcr ◽  
Hepatitis G Virus ◽  
Virus Rna ◽  
High Prevalence ◽  
Race Ethnicity ◽  
Hepatitis G ◽  
Virus C ◽  
Higher Sensitivity

The aim of this study was to investigate the presence of the Hepatitis G Virus on a population of blood donors from São Paulo, Brazil and to evaluate its association to sociodemographic variables. Two RT-PCR systems targeting the putative 5'NCR and NS3 regions were employed and the former has shown a higher sensitivity. The observed prevalence of HGV-RNA on 545 blood donors was 9.7% (CI 95% 7.4;12.5). Statistical analysis depicted an association with race/ethnicity, black and mulatto donors being more frequently infected; and also with years of education, less educated donors presenting higher prevalences. No association was observed with other sociodemographic parameters as age, gender, place of birth and of residence. DNA sequencing of nine randomly chosen isolates demonstrated the presence of genotypes 1, 2 and 3 among our population but clustering of these Brazilian isolates was not detected upon phylogenetic analysis.

scholarly journals Sensitive detection and quantification of SARS-CoV-2 in saliva

2020 ◽  
Author(s):  
Mustafa Fatih Abasiyanik ◽  
Blake Flood ◽  
Jing Lin ◽  
Sefika Ozcan ◽  
Sherin J Rouhani ◽  
...  
Keyword(s):  
Nasal Swab ◽  
Digital Pcr ◽  
Rt Pcr ◽  
Community Screening ◽  
Viral Loads ◽  
Reverse Transcription Pcr ◽  
Testing Facility ◽  
Wide Range ◽  
Nasal Swabs ◽  
Higher Sensitivity

AbstractSaliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

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