Use of nanotechnology for infectious disease diagnostics: application in drug resistant tuberculosis

Acid Fast Bacilli (AFB) positive sputum specimens (n = 541) were collected from the Central Chest Clinic, Colombo from 2013 to 2016. The sputum specimens were processed and cultured on Lowenstein Jensen medium to isolate MTb strains. A total of 450 cultures were confirmed as MTb using phenotypical characters (growth rate, color and colony morphology) and PCR amplification of IS6110 insertion element of the MTb genome. Fifteen (15) INH resistant isolates were identified among the 450 isolates by the agar proportion method. In addition, 41 INH resistant MTb strains isolated from sputum samples received from chest clinics were directly collected from National Tuberculosis Reference Laboratory (NTRL), Welisara, Sri Lanka.

Genomic DNA of INH resistant MTb isolates was extracted using phenol-chloroform method [21]. The extracted DNA was quantified by agarose gel electrophoresis (1.5%) using lambda DNA (539 ng/ μl, Promega).

Multiplex PCR was performed using two specific primer sets to amplify a 217 bp fragment of katG gene flanking the codon 315 and a 249 bp fragment of IS6110 insertion region specific to MTC for identification of INH resistance and MTb [22] simultaneously (Table 1). MTC consists of seven Mycobacterium species including MTb, M. africanum, M. bovis, M. microti, M. canetti, M. caprae and M. pinnipedii.

The H37Rv reference MTb strain and a known INH resistant MTb strain confirmed by the NTRL, Welisara, Sri Lanka were used as quality control strains.

The AuNP based lateral flow assay requires two types of probes; detection probes conjugated to AuNP and biotin labeled capture probes. In this study two detection probes and four capture probes were designed.

Detection probe katG (DP katG): Oligonucleotide sequence which is complementary to the 5′ end of the amplified DNA fragment (target) of katG gene.

Detection probe for TB (DP TBD): Oligonucleotide sequence which is complementary to the 5′ end of the amplified DNA fragment of IS6110 insertion region.

Capture probe for wild type and the mutated strains detection (CP WT and CP ACC): Oligonucleotide sequence which is complementary to the 3′ end of the target amplified DNA fragment of katG gene. These two probes were designed focusing the codon 315 position where the most common point mutation occurs.

These probes were designed using IDT Oligo Analyzer 3.1 tool to contain a poly A tag and thiol or biotin modifications as described in previous procedures [9, 23, 24] (Table 2). The poly A tag was introduced to the 5′ end of the detection probes and to the 3′ end of each of the capture probes. Detection probes were thiol-modified and conjugated with AuNPs under optimized conditions. Thiol modification facilitated the stronger binding of AuNP to the detection probe via the formation of strong Au-S bonds [7, 8]. Capture probes were labeled with biotin for the easy conjugation with streptavidin.

AuNPs were prepared according to the previously described citrate method [23, 25]. First, all glassware were thoroughly cleaned with aqua regia (three parts conc. HCl, one part conc. HNO₃), rinsed with copious amounts of water followed by nanopure water, and oven-dried prior to use. In a 500 mL round-bottom flask, 200 mL of 1 mM HAuCl₄ prepared in nanopure water was brought to a boil with vigorous stirring under continuous condensation. To this solution, 4 mL of 39 mM trisodium citrate was added. The solution turned deep blue within 20 s, and finally the color changed to wine-red few minutes later indicating the formation of AuNPs. Boiling was continued for an additional 10 min to ensure complete reaction and the colloid solution was then stirred for further 15 min while allowing it to cool to room temperature. The resulting AuNP suspension was aliquoted into 2 mL volumes and centrifuged at 6000 rpm for 1 h to remove any unreacted material from the supernatant. The separated AuNP pellet was then diluted using a 0.5 mM citrate storage buffer back to the initial volume (2 mL) and the AuNPs were re-dispersed in solution followed by the centrifugation at 6000 rpm for another 1 h. This step was repeated for 3 times to ensure the complete removal of unreacted materials in the as-synthesized media and hence increased the stability of AuNPs. Localized Surface Plasmon Resonance spectrum (LSPR) of purified Au-NPs indicated a λ_max of 523 nm [7] as shown in Fig. 1 (A). According to the approach previously developed by Haiss and co-workers [26], this LSPR behavior evidenced the presence of gold nanoparticles of ~ 20 nm average diameter, at a ~ 2.5 nM particle concentration. Further, the narrow spectral bandwidth and the absence of any secondary spectral features in the LSPR spectrum of the synthesized AuNP, subsequent to purification, indicated good homogeneity in particle size and appreciable stability under the experimental conditions. Additionally, high resolution transmission electron microscopic (HRTEM – JEOL JEM 2100) images of the prepared AuNPs were obtained to reconfirm the size of the AuNPs as shown in the representative HRTEM micrograph image given in Fig. 1 (B). The HRTEM indicated the presence of spherical and monodisperse gold nanoparticles of ~ 20 nm diameter, reconfirming the inference made via the LSPR spectrum. Importantly, these particles were very stable in solution and no changes could be observed in the LSPR spectra even after several weeks post-synthesis. Hence, the AuNPs were stored at 4 °C until they were utilized in the conjugation procedure.

The preparation of DNA conjugated AuNPs was modified from the procedure previously described [24]. First, 20 μL of 100 μM thiol–modified oligonucleotides were gently added to 500 μL of colloidal gold. The solution was incubated at 4 °C in the dark conditions for 24 h. Then the solution was added to 10 mM phosphate buffer (pH = 7). A 30 μL volume of 1 M NaCl solution was added drop wise to the solution and centrifuged at 12000 rpm for 20 min. The resulting conjugated AuNPs were blocked in 500 μL of 5% Bovine Serum Albumin (BSA) solution for 30 min at 25 °C followed by 12000 rpm centrifugation for 15 min. The final colloid sample was re-dispersed in 500 μL of sample eluent buffer (10% sucrose, 20 mM Na₃PO_4, 5% BSA, 0.25% Tween 20) [23]. The addition of BSA, tween 20 and sucrose facilitates the stability of AuNPs. Conformation of the conjugation of AuNPs to detection probes was done using spectrophotometric analysis [7, 8]. A shift of LSPR λ_max from 523 nm to 526 nm showed that the prepared AuNPs were conjugated with the oligonucleotides.

Conjugation of streptavidin to biotin labeled capture probes was done using the previously described procedure [23] with slight modifications. Briefly, 1 mg of streptavidin was dissolved in 900 μL of 1x PBS. A 220 μL volume of this solution was mixed with 20 μL of 1 mM capture probe in a microcentrifuge tube and incubated 1 h at 25 °C and 1 h at 37 °C. Excess probes were removed by 30,000 cut off centrifugal filter (Millipore) by centrifugation at 6000 rpm. Resulted conjugates were washed with 300 μL of 1x PBS two times in the same centrifugal filter. Remaining solution of the filter was eluted into a new microcentrifuge tube and stored at 4 °C.

Lateral flow strip was designed to contain a sample pad, test zone and an absorption pad mounted on an inert plastic backing layer. The test zone includes separate test lines for mutation probe, wild type probe, MTb identification probe and the control probe (Fig. 2). The sample pad was prepared by saturating a cellulose pad with 100 μL of sample pad saturation buffer (0.25% Triton X100, 0.05 M Tris HCl, 0.15 M NaCl, pH = 8) [23]. It was dried and stored in a dessicator. Absorption pad was also a cellulose pad which was used to facilitate the flow of solutions throughout the strip via capillary action. Test zone on the nitrocellulose membrane was used to immobilise the streptavidine conjugated biotin labelled capture probes as test lines and a control line. A 0.6 μL volume of each streptavidin conjugated capture probe was spotted as a line using a micro pipette and the membrane was incubated 16 h at 37 °C. The nitrocellulose membrane was blocked with 50 μL of membrane blocking buffer (1% sucrose, 1% BSA, 2.5% Tween 20, 0.1 M NaH₂PO₄) and incubated at 37 °C for 30 min. The sample pad, nitrocellulose membrane and the absorption pad were assembled on a plastic backing layer to form the lateral flow strip (Fig. 2).

A 5 μL volume of multiplex PCR amplified product was denatured at 99 °C for 8 min and immediately cooled in ice for 5 min. Then, 10 μL of each of the TB detection and katG detection probes were mixed with the PCR amplified, denatured targets (katG gene fragment and IS6110 insertion region fragment) and hybridized at 42 °C for 15 min in a microcentrifuge tube. The resulting hybridized mixture was introduced on to the sample pad followed by the addition of 40 μL of running buffer (1% BSA, 0.2% tween20, 0.01% SDS in 6x SSC) [23]. The capture probes on the strip were then allowed to hybridize to the above mixture at 48 °C for 15 min and were washed with 4x SSC buffer.

Concentration of the extracted target genomic DNA was determined using Quantus fluorometer (Promega). Twofold serial dilution was done for the target DNA starting with 32 pg/ μL (H37Rv strain) and 50 pg/ μL (INH resistant reference strain). Thereafter, multiplex PCR was carried out for all dilutions (five dilutions of each strain). The resulted multiplex PCR products were denatured and subjected to the above assay procedure. The strips were prepared individually for each capture probe (TBD, WT and ACC) along with the control probe. The multiplex PCR products of H37Rv strain was used to determine the limit of detection (LOD) of TBD and WT capture probes while PCR product of the reference mutant strain was used to determine the LOD of ACC capture probe.

First, specificity of the assay was performed using DNA of five different species of non-tuberculous mycobacteria (NTM) (M. abscessus, M. fortuitum, M. avium, M. smegmatis, M. kansasii). These five isolates are used as the reference NTM standards in the laboratory and have been confirmed as NTM by biochemical testing and DNA sequencing.

The specificity of the TB detection probe was determined using 10 NTM isolates (other than the 5 isolates mentioned above). These NTM isolates produced negative results for IS6110 fragment PCR amplification confirming absence of MTb. Specificity of the mutant probe and wild type probe was determined using the above 20 INH susceptible isolates and 38 INH resistant isolates respectively [14]. The H37Rv reference MTb strain as well as the confirmed INH resistant isolate by NTRL was used as the controls.

Sensitivity and the specificity of each of the three probes were statistically calculated using SPSS 18 software.