Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

Ongagna-Yhombi, Serge Y.; Corstjens, Paul L. A. M.; Geva, Eran; Abrams, William R.; Barber, Cheryl; Malamud, Daniel; Mharakurwa, Sungano

doi:10.1186/1475-2875-12-74

Cited by 22 publications

(13 citation statements)

References 33 publications

(40 reference statements)

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“…The instrument-free detection on the lateral flow dipsticks revealed a higher sensitivity than the agarose gel-based detection (Figure 2 B). This finding supports previous studies, in which the nucleic acid lateral flow immunoassay in combination with PCR, demonstrates lower or similar detection limits in comparison to gel electrophoresis analysis [ 25 , 26 ]. Moreover the use of probe during the LF-RPA reaction provides an additional starting point for polymerase elongation by functioning as primer upon binding to the target sequence.…”

Section: Resultssupporting

confidence: 92%

Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

Kersting

Rausch

Bier

et al. 2014

Malar J

232

178

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BackgroundNucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing.MethodsA specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay.ResultsThe lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined.ConclusionsCombining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.

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Section: Resultssupporting

confidence: 92%

Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

Kersting

Rausch

Bier

et al. 2014

Malar J

232

178

View full text Add to dashboard Cite

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“…The most efficient technique is PCR-based diagnosis which has produced a higher specificity and sensitivity in the identification and differentiation of malaria at the species level. PCR methods can be subdivided into nested PCR, semi-nested PCR, single step multiplex PCR, and real-time or quantitative PCR assays [ 38 , 39 ]. Among them, the simplest and least technically demanding is a loop-mediated isothermal amplification (LAMP) assay [ 40 ].…”

Section: Conventional Techniquesmentioning

confidence: 99%

The development of malaria diagnostic techniques: a review of the approaches with focus on dielectrophoretic and magnetophoretic methods

Kasetsirikul

Buranapong

Srituravanich

et al. 2016

Malar J

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The large number of deaths caused by malaria each year has increased interest in the development of effective malaria diagnoses. At the early-stage of infection, patients show non-specific symptoms or are asymptomatic, which makes it difficult for clinical diagnosis, especially in non-endemic areas. Alternative diagnostic methods that are timely and effective are required to identify infections, particularly in field settings. This article reviews conventional malaria diagnostic methods together with recently developed techniques for both malaria detection and infected erythrocyte separation. Although many alternative techniques have recently been proposed and studied, dielectrophoretic and magnetophoretic approaches are among the promising new techniques due to their high specificity for malaria parasite-infected red blood cells. The two approaches are discussed in detail, including their principles, types, applications and limitations. In addition, other recently developed techniques, such as cell deformability and morphology, are also overviewed in this article.

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“…A Harris UNI-CORE punch (Catalog #WB100029) was used to cut three 2-mm-diameter disks from each dried blood spot (DBS) containing P. falciparum 3D7 at applied concentrations of 10 0 , 10 3 , and 10 6 parasites/µL and placed in 1.5-mL polyethylene microfuge tubes. 15 Parasite DNA was extracted from the DBS using QIAamp DNA Mini Kit (QIAgen) (Catalog #51304) as per the manufacturer's instructions. Briefly, lysis buffer (180 µL) was added, and the tubes were incubated with shaking (Eppendorf Thermomixer) for 60 minutes at 85°C followed by centrifugation at 14,000 × g and the addition of Proteinase K (400 µg, Ambion Catalog #AM2548) to the lysate, which was then incubated at 56°C for 60 minutes.…”

Section: Collectorsmentioning

confidence: 99%

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

et al. 2016

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Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject’s blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200–2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

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Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

Cited by 22 publications

References 33 publications

Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

The development of malaria diagnostic techniques: a review of the approaches with focus on dielectrophoretic and magnetophoretic methods

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

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