Trypanosoma such as
Trypanosoma brucei and
Trypanosoma cruzi, the causative agents for the potentially fatal African sleeping sickness and Chagas disease, respectively, have important influence on human health. African sleeping sickness occurs mainly in sub-Saharan Africa countries where it was reported that about 10,000 people were infected in 2009 [
1,
2]. Chagas disease is found in the Americas, mainly in developing areas of Latin America [
1]. The currently accepted methods for the detection of
Trypanosoma such as microscopic examination and xenodiagnoses have poor sensitivity and are also labor-intensive and time-consuming [
1,
2]. Immunological methods such as enzyme-linked immunosorbent assay, immunochromatographic dipstick test, radioimmunosorbent assay, and immunofluorescence antibody test are rapid and sensitive but not specific [
3,
4,
5,
6,
7,
8,
9]. Molecular methods such as PCR and real-time nucleic acid sequence-based amplification are very specific but expensive and time consuming, although combination of PCR and chemiluminescence southern blot has been used to improve the sensitivity of the detection of
Trypanosoma [
10,
11,
12,
13,
14,
15,
16,
17]. These techniques are not implemented in Trypanosomiasis control programs due to the high cost of the equipment [
14,
16,
18]. None of these methods are ideal to mass screening of samples such as the onset of outbreaks, epidemiological surveys, or blood unit screening [
1], and without rapid and accurate diagnoses, treatment of the corresponding diseases is unlikely. Therefore, there is a need for an assay which can rapidly, sensitively and specifically detect
Trypanosoma without the need for specialized equipment and highly trained personnel.
Lateral flow assays are inexpensive and easy to use diagnostic methods which make them ideal for use in resource-limited areas such as those affected by
Trypanosoma [
19,
20]. While gold nanoparticles are commonly used for lateral flow assays, other particles such as liposomes [
21,
22,
23] have also been investigated to lower the limit of detection [
19]. Chemiluminescence offers a unique method of signal amplification, in which horseradish peroxidase-labeled reporter probes catalyze luminol and hydrogen peroxide to generate a signal which can be quantified by chemiluminescent readers. The incorporation of chemiluminescence onto a lateral flow assay format has previously demonstrated improved sensitivity over colloidal gold [
24]. Similarly, HRP amplification has also previously been used for chromogenic signal enhancement in a nucleic acid lateral flow assay [
25]. These results demonstrate the potential of on-membrane enzymatic amplification for enhanced signal generation. In this study, a simple and sensitive chemiluminescent lateral flow assay using a horseradish peroxidase (HRP)-labeled reporter probe has been developed for nucleic acid-based detection of
Trypanosoma mRNA sequences. The capture and reporter probes were designed to target the
Trypanosoma mRNA leader sequence, a 35 nucleotide spliced sequence present on the 5' end of all
Trypanosoma mRNA, and the poly A tail found on the 3' end, respectively [
26,
27]. We demonstrate the ability to detect sub-femtomol amounts of synthetic leader sequences representative of
Trypanosoma mRNA. The resulting chemiluminescent lateral flow assay represents an inexpensive, rapid, and sensitive method for nucleic acid detection without the need for target amplification or costly equipment.