Heat-enhancing aggregation of gold nanoparticles combined with loop-mediated isothermal amplification (HAG-LAMP) for Plasmodium falciparum detection

Highlights

• A simple and sensitive method for detecting the 18S rRNA gene ofPlasmodium falciparum based on loop-mediated isothermal amplification (LAMP) using biotinylated loop primers and visualization using colorimetric streptavidin-functionalized gold nanoparticles (SA-GNPs) is proposed.

• The positive reactions remained wine red, and the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C).

• The assay was completed within 50 min, its detection limit was 1 parasite/μL, and it was highly specific for P. falciparum.

Abstract

Malaria infection represents a major public health and economic issue that leads to morbidity and mortality globally. A highly effective and uncomplicated detection tool is required for malaria control in geographical hotspots of transmission. We developed a simple and more sensitive novel approach for the detection of the 18S rRNA gene of Plasmodium falciparum based on loop-mediated isothermal amplification (LAMP) and visualization using colorimetric, streptavidin-functionalized gold nanoparticles (SA-GNPs). Two loop primers of LAMP were biotinylated to produce biotin-containing products during amplification. After the addition of SA-GNPs, clusters of avidin-biotin complexes were established in the LAMP structure. While the positive reactions remained wine red, the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C). All steps of the assay were completed within 50 min, its detection limit was 1 parasite/μL, and it was highly specific for P. falciparum. This effortless detection system with high sensitivity and specificity could provide an alternative choice for malaria diagnostics in resource-limited regions.

Introduction

Malaria is a worldwide life-threatening disease that requires prevention and, ideally, elimination. The strategy of the World Health Organization (WHO) is to use practical prevention, rapid diagnosis, and effective treatment to reduce disease and prevent deaths from malaria. In 2019, there were an estimated 405,000 deaths caused by Plasmodium falciparum infection globally [1]. In the early stage of malaria infection, the initial symptoms (fever, headache, fatigue, and muscle pain) are consistent with other flu-like diseases and can lead to confusion and misdiagnosis. Therefore, high-quality malaria diagnosis is essential for discriminating among possible etiologies. All suspected malaria cases must be confirmed using either malaria-specific rapid diagnosis tests (RDTs) or microscopy to identify malaria species before treatment. Microscopy is considered the gold standard method for diagnosis because microscopists can easily stain and directly identify stages and species of the parasite. However, the result depends on the quality of reagents and microscope and on the experience of the microscopist [2]. To diminish these problems, RDTs for malaria have been developed to detect parasite antigens and are used throughout malaria-endemic countries. Although RDTs are rapid, inexpensive, noninvasive, and simple to perform and have high sensitivity and specificity for P. falciparum detection, they cannot detect low levels of parasitemia (≤200 parasites/μL) or distinguish P. falciparum from other Plasmodium species [[3], [4], [5]]. Nested polymerase chain reaction (nPCR) was developed in the late 1980s and is now considered another gold standard method for malaria diagnosis because it can detect as few as 5 parasites/μL of blood and differentiate all human Plasmodium species. However, the use of nPCR is not practical in field clinics of malaria-endemic areas because it requires sophisticated and well-maintained laboratory equipment and has prolonged turnaround times. The detection time may require 8−12 hours or more and includes steps of DNA extraction, amplification, and of DNA amplicon detection by electrophoresis [[6], [7], [8], [9], [10]].

A new generation of gene amplification based on isothermal amplification techniques is simpler and more rapid than conventional amplification. Among those techniques, loop-mediated isothermal amplification (LAMP) is outstanding and already has various applications in biomedical diagnostics [11]. Replication starts with DNA synthesis using Bst DNA polymerase and six primers under isothermal conditions (in the range of 65 °C). The six primers consisted of the forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), backward outer primer (B3c), forward loop primer (LF) and backward loop primer (LB), which bound to 6 distinct regions on the DNA target. They amplify by strand displacement activity, producing up to 10¹⁰ DNA copies within 30−60 min [9,12,13]. Interestingly, the LAMP assay could be considered a point-of-care test, as a large number of amplicons can be isothermally amplified within an hour without the use of sophisticated equipment [[14], [15], [16]]. The large amounts of DNA amplicons and pyrophosphate ions lead to a white precipitate of magnesium pyrophosphate observable by the naked eye in the reaction tube and correlates with the amount of synthesized DNA [17]. However, if the levels of DNA amplicons are low, it is difficult to detect turbidity changes in the reaction mixture due to interobserver differences [18]. Moreover, LAMP amplicons need to be confirmed by agarose gel electrophoresis, which requires a labor-intensive and skill-dependent readout process. Alternately, to avoid electrophoresis, the LAMP products can be traced with functionalized gold nanoparticles (GNPs) and analyzed by colorimetric detection [[19], [20], [21], [22], [23]]. GNPs can be conjugated to allow amplification of readout signals using various detection strategies [24]. GNPs exhibit surface plasmon resonance [22], that is, the fluctuation and interaction of electrons between charges at the particle surface, which gives rise to a sharp and intense absorption band in the visible range. The aggregation of GNPs through interaction between specific sites leads to a change in color from red to blue, which can be clearly observed by the naked eye. The color change can be attributed to the size and clustering of these particles [[25], [26], [27], [28]]. These results can be used to confirm LAMP products in a field setting.

Based on the advantages of GNPs, providing a rapid, convenient and on-site readout technique for LAMP, we aimed to develop a sensing material for LAMP product analysis through aggregation of GNPs (GNP-LAMP). For malaria detection, six LAMP primers amplified the 18S ribosomal RNA (18S rRNA) gene of P. falciparum [[29], [30], [31]]. This is a multicopy gene located within strongly conserved regions and without cross-reaction to sequences from other pathogens. With this gene, parasite DNA can be easily amplified so that highly sensitive detection of human malaria parasites can be achieved. Moreover, the GNP-LAMP method has been reported previously with some modifications [32]. The accelerated primers, i.e., LF and LB primers, were conjugated with biotin at the 5′ end, and the GNPs were functionalized with streptavidin (SA). In our study, LAMP products of the 18S rRNA gene of P. falciparum were added to SA-functionalized GNPs (SA-GNPs) and hydrochloric acid (HCl), and aggregation was induced by heating. The biotin-containing amplicons can bind SA-GNPs and form a cluster based on the formation of avidin-biotin complexes, resulting in no free SA-GNPs in the solution. In the case of no LAMP amplicon, HCl causes free SA-GNP aggregation, leading to the formation of sediment at the bottom of the tube that can be observed by the naked eye because the addition of salt into the GNP solution shields the surface charge of these GNPs, decreases interparticle distances, and leads to particle aggregation [33]. Moreover, the kinetic energy obtained by temperature can cause aggregation due to the bleaching of stabilizers [34]. The method was developed, and its performance was examined in blood samples. The results of LAMP were compared with nPCR, and the assay was evaluated for sensitivity, specificity and detection limit.