SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination

PloS One
Weiping XuAndreas Mårtensson

Abstract

A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the referen...Continue Reading

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Citations

Apr 29, 2015·Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases·Ulrika MorrisAndreas Mårtensson
Jul 19, 2015·Parasites & Vectors·Jeffrey A BellVasyl V Tkach
Apr 22, 2017·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·Anders BjörkmanAndreas Mårtensson
Feb 7, 2019·Memórias do Instituto Oswaldo Cruz·Natália Ketrin Almeida-de-OliveiraMaria de Fátima Ferreira-da-Cruz
May 10, 2020·Malaria Journal·Fiona R P BhondoekhanUNKNOWN Southern and Central Africa International Centers of Excellence for Malaria Research
Jul 6, 2019·PloS One·Christel Gill HaanshuusStein Christian Mohn
Dec 21, 2017·PLoS Neglected Tropical Diseases·Moritoshi IwagamiShigeyuki Kano
Apr 9, 2019·Journal of Parasitic Diseases : Official Organ of the Indian Society for Parasitology·Sothorn AnucherngchaiJong-Yil Chai
May 6, 2020·Nature Communications·Jeremy K HerrenSteven P Sinkins
May 1, 2021·Diagnostics·Saiful Arefeen SazedMohammad Shafiul Alam
Jul 6, 2021·Frontiers in Cellular and Infection Microbiology·Alba Marina GimenezDaniel Youssef Bargieri

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Datasets Mentioned

BETA
AB182497.1

Methods Mentioned

BETA
PCR
electrophoresis
PCRs
dot-blot

Software Mentioned

STATA
Primer3
Primer
blast
ClustalW2

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