Before commencement of the study, ethics approval was obtained from the Ethics Committee of the Faculty of Medicine Siriraj Hospital, Mahidol University (approval number, Si129/2016). The research also fully complied with the ethics principles and guidelines for human experimentation issued by the National Research Council of Thailand. The formal consent of the participants was obtained verbally.
Extraction of DNA from blood samples
DNA was extracted from 50 µL of ethylenediaminetetraacetic acid blood samples using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s protocol. Following extraction, the DNA was eluted in 100 µL of elution buffer and stored at -20º C until use. The DNA concentration was determined using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc, Waltham, Mass., USA) before the DNA was subjected to PCR and miniPCR analysis.
Development of a miniPCR-DLFD
Primer design and optimization of standard PCR condition
The primer designs were based on alignments of the HhaI and SspI repetitive non-coding DNA sequences of B. malayi and W. bancrofti, respectively [30,31,32]. These regions contain species-specific sequences that are useful for identifying B. malayi and W. bancrofti. The HhaI and SspI sequences were obtained from the National Center for Biotechnology Information database (GenBank accession numbers M12691.1 for B. malayi and L20344.1 for W. bancrofti). Fig. 1 presents the sequences of the forward and reverse primers. A BLAST analysis (National Center for Biotechnology Information, Bethesda, Md., USA) was performed to check the specificity of the primers to the target sequences. The presence of the 145-bp and 182-bp amplification products of HhaI and SspI, respectively, was verified by 2% agarose gel electrophoresis.
The PCR amplifications were carried out in a volume of 20 µl, consisting of 10 µl of PCR master mix (Quantabio; Qiagen Beverly, LLC., Mass., USA); 0.2 µM of each forward and reverse primer; 7.2 µl of dH2O; and 2 µl of DNA template. Nuclease-free water was used as a negative control. DNA from the blood samples containing mf of B. malayi and W. bancrofti were used as positive controls.
To optimize the PCR conditions, a gradient PCR was performed using the Veriti 96-Well Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific Inc, Waltham, Mass., USA) with the annealing temperature ranging from 55–62º C for both sets of primers. At an annealing temperature of 56° C, both set of primers (HhaI and SspI) amplified clearly detectable products. The optimized amplification conditions for both primer sets included an activation step at 95° C for 5 min, followed by a 30-step amplification of 30 s at 95° C, 30 s at 56° C, and 30 s at 70° C, with a last step at 70° C for 5 min.
Assay conditions to validate the miniPCR
The PCR assays for B. malayi and W. bancrofti were separately performed using a miniPCR instrument (DBA miniPCR bio; Amplyus LLC., Cambridge, Mass., USA), employing the same reagents and conditions used for the standard PCR, as noted above. For use in a DLFD, the 5’ ends of the designed forward primers of HhaI and SspI were labelled with fluorescein isothiocyanate (FITC). The 5’ ends of reverse primers of HhaI and SspI were labelled with digoxin (DIG) and biotin, respectively. The sequences of the primers and their corresponding amplicons are presented in Fig. 1.
Construction of DLFD for nucleic acid detection
A DLFD is composed of four parts: the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad. The sample pad is pretreated with buffer, and it can offer suitable pH and ion strength for the detection. The conjugate pad is used for the storage of reporter molecules (colloidal gold conjugated mouse anti-FITC). For the development of our DLFD, anti-digoxin (anti-DIG; Test line 1) and streptavidin (Test line 2) were sprayed on the nitrocellulose membrane to create test zones using the IsoFlow Reagent Dispenser (Imagene Technology, Inc., Hanover, N.H., USA), whereas anti-mouse antibody was sprayed on the nitrocellulose membrane to form a control zone (control line) by the AirJet Quanti 3000 Nanoliter aerosol dispenser (BioDot, Inc., Irvine, Calif., USA). The membrane was then dried at 37° C for 12 hours. The nitrocellulose membrane was attached to the central part of an adhesive plate. The lateral flow dipstick was then assembled, as shown in Fig 2; a schematic illustration of the DLFD is also presented in that figure.
Optimization of anti-DIG and streptavidin concentration
To optimize the concentration of anti-DIG, various concentrations of anti-DIG (0.5 µg/strip, 0.75 µg/strip or 1 µg/strip; Test line 1) were sprayed on the nitrocellulose membrane to optimize the concentration of anti-DIG, using the IsoFlow Reagent Dispenser (Imagene Technology, Inc.). The concentrations were at 1 µl per mm. The lateral flow dipstick was then assembled. Various concentrations of streptavidin (i.e., 0.5 µg/strip, 0.75 µg/strip, or 1 µg/strip) (Test line 2) were sprayed on the nitrocellulose membrane to optimize concentration of streptavidin using the AirJet Nanoliter aerosol dispenser (BioDot, Inc.). The assay was performed using positive (DNA of B. malayi) and negative (dH2O) controls.
DLFD for detection of amplification products
To detect the amplification products of each sample, 1 µl of amplification product from each set of primers (specific to HhaI and SspI) was added into a well of the 96-well plate containing 100 µl of sample buffer. The dipstick was placed into the well vertically, and the reaction was read within 5–10 min. The appearance of positive, pink-coloured lines was observed using the naked eye on both the test and control lines. With respect to negative results, the pink-coloured line was apparent solely on the control line.
The miniPCR-DLFD specificity
To verify the specificity of the miniPCR-DLFD-based detection platform, genomic DNA samples isolated from 11 other parasites—Trichinella spiralis, Angiostrongylus cantonensis, Gnathostoma spinigerum, Enterobius vermicularis, Necator americanus, Taenia solium, Plasmodium falciparum, Litomosoides sigmodontis, Brugia pahangi, Dirofilaria immitis and D. repens—were used to measure off-target PCR amplification by the miniPCR-DLFD. The amount of DNA used for the miniPCR of each parasite was 20 ng/reaction.
Detection limit of miniPCR-DLFD
To study the detection limit of the miniPCR-DLFD, mf of B. malayi was spiked into ethylenediaminetetraacetic acid blood specimens obtained from healthy subject, as listed in Table 1. Four sets of the samples were prepared (sample IDs 1–24). The DNA of B. malayi as well as negative blood samples were used as positive and negative controls. DNA that had been extracted from the blood samples underwent amplification using the standard PCR as well as the miniPCR, followed by DLFD assay.
Comparison of the Giemsa-stained thick blood smears, standard PCR based amplification and the miniPCR-DLFD assay
The study population consisted of 10 subjects positive for B. malayi mf, 14 subjects positive for W. bancrofti mf, and 50 healthy controls. Blood was also collected from the 14 W. bancrofti mf positive subjects on day 7 following treatment with diethylcarbamazine and albendazole treatment (Table 2). Giemsa staining of thick blood smears, standard PCR amplification, and miniPCR-DLFD assays were performed.
For field validation of the miniPCR-DLFD, a total of 216 blood samples were collected from Thai residents in Narathiwat Province, an area endemic for brugian filariasis. A further 328 blood samples were obtained from Burmese immigrants who resided along the Thailand–Myanmar border in Tak Province, which is a region endemic for W. bancrofti (Table 2). All of the blood samples from these study participants were collected during the daytime.
Microfluidic device coupled with miniPCR-DLFD
Detection of microfilariae was undertaken in all study samples using the microfluidic device, as described . Briefly, 50 μl of blood was suspended in 150 μl of lysis buffer A, mixed, and then incubated at room temperature for 10 min. The sample was uploaded for analysis by drawing buffer B, 15 μl, into a one ml syringe that connects to the adapter and to the inlet port. The pump was started and the sample solution introduced into the microfluidic device via the inlet port. The microfilariae were trapped in the microfluidic chip while the remaining solution exited the microfluidic chip via the outlet port into the waste tube. The trapped microfilariae were inspected by light microscopy at 10x magnification. To identify species of the trapped mf in the microfluidic chips, 100 μl Tris-EDTA buffer was dispensed into the chip via the inlet port. The chip was placed on a hot plate at 56°C for 15 min, after which the solution containing the trapped mf was withdrawn through the outlet port and transferred to a 1.5 ml Eppendorf tube. The tube was subjected to centrifugation at 15,520× g for 10 min. The supernatant was discarded, and DNA was extracted from the pelleted material using the Roche high pure PCR template preparation kit, according to the manufacturer’s instructions (Roche Diagnostics GmbH, Penzberg, Germany). Species identification was performed by HhaI/SspI gene amplification by miniPCR (Amplyus) followed by use of the DLFD testing using the above-mentioned miniPCR assay primers and employing the same reaction recipes and conditions as described above.