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Assessment of malaria real-time PCR methods and application with focus on low-level parasitaemia
- Source :
- PLoS ONE, PLoS ONE, Vol 14, Iss 7, p e0218982 (2019)
- Publication Year :
- 2018
-
Abstract
- In epidemiological surveys and surveillance the application of molecular tools is essential in detecting submicroscopic malaria. A genus-specific conventional cytochrome b (cytb) PCR has shown high sensitivity in field studies, detecting 70% submicroscopic malaria. The main objective of this study was to assess the conversion from conventional to real-time PCR testing both SYBR and probe protocols, and including quantitative (q) PCR. The protocols were assessed applying well-defined clinical patient material consisting of 33 positive and 80 negative samples. Sequencing of positive PCR products was performed. In addition, a sensitivity comparison of real-time PCR methods was done by including five relevant assays investigating the effect of amplification target and platform. Sensitivity was further examined using field material consisting of 111 P.falciparum positive samples from Tanzanian children (< 5 years), as well as using related patient data to assess the application of q-PCR with focus on low-level parasitaemia. Both the cytb SYBR and probe PCR protocols showed as high sensitivity and specificity as their conventional counterpart, except missing one P. malariae sample. The SYBR protocol was more sensitive and specific than using probe. Overall, choice of amplification target applied is relevant for achieving ultra-sensitivity, and using intercalating fluorescence dye rather than labelled hydrolysis probes is favourable. Application of q-PCR analysis in field projects is important for the awareness and understanding of low-level parasitaemia. For use in clinical diagnosis and epidemiological studies the highly sensitive and user-friendly cytb SYBR q-PCR method is a relevant tool. The genus-specific method has the advantage that species identification by sequencing can be performed as an alternative to species-specific PCR. publishedVersion
- Subjects :
- 0301 basic medicine
Plasmodium
Focus (geometry)
Physiology
Pcr cloning
Genes, Protozoan
Artificial Gene Amplification and Extension
Parasitemia
Polymerase Chain Reaction
law.invention
0302 clinical medicine
Filter Paper
law
Medicine and Health Sciences
Organic Chemicals
Polymerase chain reaction
Protozoans
Multidisciplinary
Malarial Parasites
Eukaryota
Drugs
Cytochromes b
Body Fluids
Laboratory Equipment
Real-time polymerase chain reaction
Blood
Child, Preschool
Quinolines
Medicine
Engineering and Technology
Anatomy
Research Article
Science
030106 microbiology
030231 tropical medicine
Plasmodium falciparum
Equipment
Computational biology
Biology
Diamines
Real-Time Polymerase Chain Reaction
Research and Analysis Methods
Sensitivity and Specificity
03 medical and health sciences
Antimalarials
Parasite Groups
medicine
Parasitic Diseases
Humans
Benzothiazoles
Molecular Biology Techniques
Molecular Biology
Pharmacology
Infant, Newborn
Organisms
Infant
Reproducibility of Results
Biology and Life Sciences
Patient data
DNA, Protozoan
medicine.disease
Tropical Diseases
Parasitic Protozoans
Highly sensitive
Malaria
Clinical diagnosis
Parasitology
Apicomplexa
Subjects
Details
- ISSN :
- 19326203
- Volume :
- 14
- Issue :
- 7
- Database :
- OpenAIRE
- Journal :
- PloS one
- Accession number :
- edsair.doi.dedup.....2dff30b3bc353530cea7e9c83ef47389