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

Abstract

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.

Keywords: Plasmodium falciparum; Plasmodium vivax; loop-mediated isothermal amplification (LAMP); malaria; molecular detection; point-of-care (POC).

Figure 1

LAMP of DNA isolated from DBS showing real-time amplification in the positive control (genomic Plasmodium DNA) and amplification from DNA isolated from DBS containing 10³ and 10⁶ parasites/µL.

Figure 2

(A) Direct LAMP of MPB without prior DNA isolation. Serial dilution of blood samples from 1:1,000 to 10,000 dilutions allowed following the amplification with real-time detection of fluorescence. However, smaller dilutions of blood (ie, 1× and 1:10) showed an apparent lack of amplification in real time. It should be noted that a dilution of 1:100 was not consistently sufficient to allow real-time detection depending on the blood sample. (B) Image of the agarose gel electrophoresis of the samples amplified in (A). All of the samples showed the characteristic ladder pattern for LAMP after isothermal amplification.

Figure 3

(A) LAMP as a function of concentration of genomic DNA sample MRA-731G. (B) Graph of the resulting C_t values as a function of MRA-731 DNA concentration (ng/µL) (n = 7). (C) Annealing curves of the genomic sample MRA-731 for each dilution yielded a single amplicon product.

Figure 4

LAMP curves for a serial dilution of MPB blood (MRA-731) from 1:1,000 to 10,000,000.

Figure 5

(A) LAMP of MPB treated with agents indicating that BSA facilitates amplification more efficiently than other agents when heated at 70°C for 15 minutes. (B) Image of the electrophoresis gel of the LAMP amplified treated MPB.

Figure 6

Effect of temperature on the LAMP cross-over threshold (C_t). All incubations were for 5 minutes at the indicated temperature, followed by a 4-µL aliquot amplified using LAMP.

Figure 7

(A) Mobicol column with loaded Copan collector head. (B) Mobicol assembly post-centrifugation with eluted sample after centrifugation from the Copan collector head suspended in Mobicol column inserted in the 1.5-mL Eppendorf centrifuge tube.

Figure 8

LAMP of spiked WMSS samples recovered from Hydraflock and Copan collectors.

Figure 9

(A) LAMP detection of P. falciparum DNA using LAMP primers targeted to the parasite’s mitochondrial cytochrome oxidase subunit 1 gene. The P. falciparum-specific primers did not amplify two different samples of P. vivax DNA. (B) LAMP detects both P. falciparum and P. vivax DNA when targeting the 18S rRNA gene present in all Plasmodium species.