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

ABSTRACT: 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.

SUBMITTER: Ferreira B

PROVIDER: S-EPMC8608302 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. RESULTS: The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro. CONCLUSION: Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons.

Project description:BACKGROUND:Candida species are considered as the cause of one of the most important opportunistic fungal diseases. Accurate identification of Candida species is important because of antifungal susceptibility patterns are different among these species, so proper identification helps in the selection of antifungal drugs for the prevention and treatment. Phenotypic methods for identification of Candida species, which are widely used in clinical microbiology laboratories, have some limitations. Real-time PCR followed by the high-resolution melting analysis (HRMA) is a novel approach for the rapid recognition of pathogenic fungi. Molecular phylogeny is essential for obtaining a better understanding of the evolution of the genus Candida and the identification of the relative degree of the Candida species. The purpose of this study was molecular identification of Candida isolates by Real-time PCR-high-resolution melting analysis and investigation of the genetic diversity of Candida species. METHODS:Two hundred and thirty-two Candida isolates including 111 Candida isolates obtained from 96 HIV/AIDS patients and 121 Candida isolates obtained from 98 non-HIV persons were identified by real-time PCR and high-resolution melting curve analysis. To evaluate genetic diversity and relationships among Candida species, PCR products of nine clinical Candida isolates, as a representative of each kind of species, were randomly selected for DNA sequence analysis. RESULTS:In HIV/AIDS patients, six species of Candida spp. were identified as follows: C albicans (n = 64; 57.7%), C glabrata (n = 31; 27.92%), C parapsilosis (n = 9; 8.1%), C tropicalis (n = 4; 3.6%), C krusei (n = 2; 1.8%), and C kefyr (n = 1; 0.90%). In non-HIV persons, we identified eight species of Candida including C albicans (n = 46; 38.33%) followed by C glabrata and C krusei (each one, n = 18; 15%), C tropicalis (n = 13; 10.83%), C lusitaniae (n = 12; 5.17%), C parapsilosis (n = 10; 4.31%), and C kefyr and C guillermondii (each one, n = 2; 1.66%). Also, the phylogenetic analysis showed the presence of two main clades and six separate subclades. Accordingly, about 88.9% of the isolates were located in clade I and 11.10% of the studied isolates were in clade II. CONCLUSIONS:Real-time PCR followed by high-resolution melting analysis (HRMA) is known as a reliable, fast, and simple approach for detection and accurate identification of Candida species, especially in clinical samples.

Project description:BackgroundBabesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp.MethodsUsing 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR.ResultsThe LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA qPCR positive.ConclusionsWe have developed a more sensitive qPCR assay with a more expansive range of Babesia spp. detection by targeting a highly conserved region of mtDNA, when compared to an established 18S qPCR.

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

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