ShDraI-RPA modifications and optimisation

Modified oligonucleotides

RPA primers and probe targeting the S. haematobium Dra I tandem repeat region (GenBank accession number: DQ157698.1) described by Rostron et al.²³ were modified with the aim of increasing assay specificity, whilst maintaining high levels of sensitivity. Modifications included the addition of a phosphorothioate backbone (denoted as “s” in the primer sequence) before the last 3’ base of the reverse primer, and inversion of the position of the fluorophore 6-carboxyfluorescein (6FAM) and the black-hole quencher 1 (BHQ1) within the fluorescent probe (Table 3).

RPA reaction and visualisation of the results

ShDraI-RPA reactions were performed using the TwistAmp Exo Kit (TwistDx, Cambridge, UK) following the manufacturer instructions and as described by Rostron et al.²³. All reactions were performed in triplicate, and positive (100 pg of S. haematobium DNA) and negative (nuclease-free water) controls were included with each RPA run of 16 reactions. RPA reaction tubes were incubated at 42 °C for 20 min in the portable fluorometer Axxin T16-ISO (Axxin Diagnostics, Australia) where the fluorescence was measured, and results were observed in real-time via T16-ISO Software (V2.1.0.3- Axxin Diagnostics, Australia). Samples were considered positive for S. haematobium when an increase of ≥ 500 relative units of fluorescence (RFU) above the baseline was observed²⁷. Additionally, to verify whether the assay could provide a qualitative binary diagnosis without the need for a fluorometer, the end-point fluorescence was observed by exposing the reaction tubes to a source of blue light after the amplification. The blue light has a wavelength of approximately 450 to 495 nm and the absorbance of 6FAM is 493 nm. This means that in positive samples (i.e. which contain amplified DNA), where the fluorophore, 6FAM, has been released from the quencher, is able to absorb the blue light and produce a fluorescence that is visible to the naked eye.

Source of biological samples

Genomic DNA (gDNA) isolated from S. haematobium and other Schistosoma species (S. mansoni, S. curassoni, S. bovis, S. rodhaini, S. mattheei, S. japonicum, S. margrebowiei and S. guineensis) were obtained from the Schistosomiasis Collection at The Natural History Museum- UK (SCAN)⁴³ repository. The Medical Malacology Collection at René Rachou Institute, Fiocruz Minas- Brazil (Fiocruz-CMM) provided samples from trematodes belonging to the families Clinostomidae, Echinostomatidae, Fasciolidae, Notocotylidae, Spirorchiidae and Strigeidae, and SCAN provided trematodes belonging to the genus Tricobilharzia. Genomic DNA from helminths of medical importance (i.e. Ascaris lumbricoides, Enterobius vermicularis, Trichuris trichiura, and hookworms) were provided by the Helminthology and Medical Malacology Laboratory from the René Rachou Institute, Fiocruz Minas- Brazil. To test for Gram-negative bacteria which commonly cause UTIs, gDNA previously obtained by Rosser et al.²⁰from Escherichia coli and Klebsiella pneumoniae were used. In addition, Charing Cross Hospital (London- UK) supplied a reference colony of Proteus mirabilis (ATCC strain 35659; https://www.atcc.org/products/35659) from their microbiology laboratory. Single S. haematobium ova were provided frozen by the Snail Schistosome Resource (SSR, Natural History Museum, UK) via the NIAID Schistosomiasis Resource Centre (SRC, Biomedical Research Institute, USA). All DNA samples were stored at -20 °C before use.

Analytical specificity testing

Genomic DNA was isolated from all organisms listed above using the DNeasy Blood & Tissue (Qiagen GmbH, Germany) kit, according to the manufacturer’s instructions. All gDNA isolates were quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and normalised to a working concentration of 100 pg/µL using nuclease-free water. All samples were analysed using the modified ShDraI-RPA oligonucleotides using various incubation temperature (39, 40 and 42 °C) to determine the optimal temperature for the assay specificity.

Analytical sensitivity testing

Genomic DNA isolated from adult worms of S. haematobium was used to obtain tenfold serial dilutions from 1 ng/µL to 1 fg/µL. Synthetic copies of the Dra I target region were obtained as described by Donnelly et al.²⁷ and dilutions ranging from 1 × 10⁵ to 1 × 10⁰ copies/µL were prepared. Genomic DNA from single eggs was isolated using the SwiftX DNA extraction kit (Xpedite Diagnostics GmbH, Germany) following the manufacturer’s instruction (protocol 1 from the handbook). All DNA (genomic and synthetic) samples were analysed using the modified ShDraI-RPA, as described above, to determine assay sensitivity. The direct addition of single S. haematobium eggs (i.e. without their gDNA extracted) to the RPA reactions was also tested.

Spiked urine samples

Aliquots (100 µL) of urine from a donor (from a non-endemic country) were spiked with: (i) a single egg of S. haematobium and, (ii) 1 ng of S. haematobium gDNA. Also, non-spiked aliquots were processed as negative controls.

All of the spiked and non-spiked aliquots were processed and used as follows: (i) direct addition to the RPA reaction; (ii) DNA extraction using DNeasy Blood & Tissue kit (Qiagen), following the manufacturer’s protocol; (iii) DNA extraction using a rapid, low resource method for POC use, SwiftX DNA kit (Xpedite), according to the manufacturer’s protocols one and two, testing variations on the time and temperature of lysis incubation.

Briefly, the SwiftX DNA kit protocol one (hereon, the short protocol) consists of the addition of 100 µL of the lysis buffer DL and 30 µL of Beads A to the sample (100 µL). The mixture was homogenised for 10 s by vortexing and incubated at 95 °C (or alternatively, at room temperature- RT) for five or 15 min. The tubes were placed in a magnetic rack for one minute and the supernatant (containing the gDNA) was transferred to a fresh 1.5 mL microcentrifuge tube and stored at −20 °C until use. Thus, four variations of the short protocol were tested: (i) lysis incubation at 95 °C for 5 min; (ii) lysis incubation at 95 °C for 15 min; (iii) lysis incubation at RT for five min; (iv) lysis incubation at RT for 15 min.

The SwiftX DNA kit protocol two (hereon, the long protocol) consists of the addition of 100 µL of the buffer EN and 30 µL of Beads A to the sample (100 µL). The mixture was homogenised for 10 s by vortexing and incubated at RT for 3 min. The tubes were then placed in a magnetic stand for 1 min and the supernatant was discarded. 100 µL of the lysis buffer DN was added to each tube that was subsequently homogenised by vortexing. The mixture was incubated at 95 °C (or alternatively, at RT) for five or 15 min and then placed into a magnetic stand for 1 min. The supernatant (containing the gDNA) was transferred to a fresh 1.5 mL microcentrifuge tube and stored at -20 °C until use. Again, four variations of the long protocol were tested: (i) lysis incubation at 95 °C for 5 min; (ii) lysis incubation at 95 °C for 15 min; (iii) lysis incubation at RT for 5 min; (iv) lysis incubation at RT for 15 min.

In total, eight protocols were tested for the SwiftX DNA kit. The concentration of gDNA in all the extracts was measured using a Nanodrop™ spectrophotometer.

qPCR analysis

DNA isolated from all egg- and gDNA-spiked urine samples extracted using the different methods were analysed by qPCR. This was performed as a confirmatory test for the isolation of S. haematobium DNA from each sample type by the different methods tested^27,44,45. The quantitative nature of this assay also allowed comparison of the DNA yield of the different sample preparation methodologies described above. Schistosoma genus-specific oligonucleotides targeting the internal transcribed spacer region 2 (ITS 2) were used in a 20 µl reaction containing 0.3 µM of each primer (Forward- Ssp48F: 5’-GGTCTAGATGACTTGATYGAGATGCT- 3’; Reverse- Ssp124R: 5’-TCCCGAGCGYGTATAATGTCATTA-3’), 0.1 µM of the probe (Ssp78T: 5’- FAM-TGGGTTGTG/ZEN/CTCGAGTCGTGGC-IowaBlack-FQ -3’), 1× Luna Universal Probe qPCR MasterMix (New England Biolabs), and 1 µl of template DNA. The StepOne Plus thermocycler (Applied Biosystems) was used with the following cycling conditions: 2 min initial denaturation at 95 °C, and 50 cycles of 95 °C denaturation for 15 s followed by combined 60 °C annealing/extension for 60 s. Samples were considered positive when the cycle threshold (Ct) value was < 38. This step was particularly important for assessing the different sample preparation methodologies.

Assay stability and longevity

To determine the stability of the ShDraI-RPA reagents (i.e. primers, probe, and components from the TwistAmp exo kit- RPA pellet, RPA rehydration buffer and magnesium acetate MgAc) at ambient temperature in schistosomiasis-endemic settings, all reagents were stored at ± 27 °C in a dark environment without the addition of any stabilizers. The reagents were then used to perform ShDraI-RPA reactions on days: 0, 1, 2, 3, 7, 14, 21, 30, 60 and 90. The ShDraI-RPA reactions were set up as described above and positive (100 pg of S. haematobium DNA) and negative (nuclease-free water) controls were included in all tests. Results were observed in real time via the Axxin T16- ISO portable fluorometer and software, and post-amplification exposure of the tubes to a blue light.