scholarly journals Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260087
Author(s):  
Beatriz Iandra da Silva Ferreira ◽  
Natália Lins da Silva-Gomes ◽  
Wagner Luis da Costa Nunes Pimentel Coelho ◽  
Vanessa Duarte da Costa ◽  
Vanessa Cristine de Souza Carneiro ◽  
...  

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

2021 ◽  
Author(s):  
Beatriz Iandra da Silva Ferreira ◽  
Natalia Lins da Silva Gomes ◽  
Wagner Luis da Costa Nunes Pimentel Coelho ◽  
Vanessa Duarte Costa ◽  
Vanessa Cristine de Souza Carneiro ◽  
...  

With the emergence of the Covid-19 pandemic, the world faced an unprecedented need for RT-qPCR-based molecular diagnostic tests, leading to a lack of kits and inputs, especially in developing countries. Hence, the costs for commercial kits and inputs were overrated, stimulating the development of alternative methods to detect SARS-CoV-2 in clinical specimens. The availability of the complete SARS-CoV-2 genome at the beginning of the pandemic facilitated the development of specific primers and standardized laboratory protocols for Covid-19 molecular diagnostic. High-sensitive and cost-effective molecular biology technique based on the Melting Temperature differences between purine and pyrimidine bases can be used to the detection and genotyping of pathogens in clinical specimens. Here, a RT-qPCR assays with High Resolution Melting (HRM-RTqPCR) was developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states in Brazil, in comparison to a commercial TaqMan RT-qPCR assay, as gold standard. The sensitivity of the HRM-RTqPCR assays targeting N, RdRp and E were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 targets, and diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, the HRM-RTqPCR emerges as an alternative and low-cost methodology to increase the molecular diagnostic of patients suspicious for Covid-19, especially in restricted-budget laboratories.


2017 ◽  
Vol 21 (2) ◽  
pp. 66-73
Author(s):  
Hema L. Ramkumar ◽  
Harini V. Gudiseva ◽  
Kameron T. Kishaba ◽  
John J. Suk ◽  
Rohan Verma ◽  
...  

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89395 ◽  
Author(s):  
Jiyoun Yeo ◽  
Erin L. Crawford ◽  
Thomas M. Blomquist ◽  
Lauren M. Stanoszek ◽  
Rachel E. Dannemiller ◽  
...  

2016 ◽  
Vol 54 (11) ◽  
pp. 2655-2660 ◽  
Author(s):  
Margaret V. Powers-Fletcher ◽  
Kimberly E. Hanson

The direct detection ofAspergillusnucleic acid in clinical specimens has the potential to improve the diagnosis of aspergillosis by offering more rapid and sensitive identification of invasive infections than is possible with traditional techniques, such as culture or histopathology. Molecular tests forAspergillushave been limited historically by lack of standardization and variable sensitivities and specificities. Recent efforts have been directed at addressing these limitations and optimizing assay performance using a variety of specimen types. This review provides a summary of standardization efforts and outlines the complexities of molecular testing forAspergillusin clinical mycology.


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