Dataset Information

Molecular differentiation of Schistosoma japonicum and Schistosoma mekongi by real-time PCR with high resolution melting analysis.

ABSTRACT: Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07? and 85.7±0.07?, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

SUBMITTER: Kongklieng A

PROVIDER: S-EPMC3916453 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Molecular differentiation of Schistosoma japonicum and Schistosoma mekongi by real-time PCR with high resolution melting analysis.

Kongklieng Amornmas A Kaewkong Worasak W Intapan Pewpan M PM Sanpool Oranuch O Janwan Penchom P Thanchomnang Tongjit T Lulitanond Viraphong V Sri-Aroon Pusadee P Limpanont Yanin Y Maleewong Wanchai W

The Korean journal of parasitology 20131231 6

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to ...[more]

PMID: 24516269

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Project description: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.

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.

Dataset Information

Molecular differentiation of Schistosoma japonicum and Schistosoma mekongi by real-time PCR with high resolution melting analysis.

Publications

Molecular differentiation of Schistosoma japonicum and Schistosoma mekongi by real-time PCR with high resolution melting analysis.

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