Molecular methods for detecting drug resistance

All the molecular methods were performed using either DNA extracted from clinical specimens with the commercial Maxwell® 16 Viral Total Nucleic Acid Purification Kit (Promega, USA) (Article III), or crude DNA extracts obtained from clinical specimens or clinical strains as previously described [62]. Briefly, few colonies of clinical strains cultured in solid media were resuspended in 300 µl of molecular biology-grade water; or a 1 ml aliquot of the decontaminated specimen was centrifuged at 10,000 x g for 15 min, the supernatant was discarded, and the pellet was resuspended in 100 µl of molecular biology-grade water. These preparations of clinical strains and specimens were then incubated at 95°C for 20 min, sonicated for 15 min, and centrifuged at 22,000 x g for 5 min, and the supernatant was collected. DNA extracts were stored at -20ºC until use.

4.3.1. Reverse hybridization array

The reverse hybridization array evaluated (GenoFlow DR-MTB Array Test, DiagCor Bioscience, Hong Kong) is aimed to detect mutations in rpoB, katG, and inhA promoter associated with resistance to RIF and INH (Article I). The method is based on PCR amplification and reverse hybridization on arrays using the FT-PRO flow-through system.

The test was performed following the manufacturer’s instructions. Briefly, the PCR reaction was prepared with 19.6 µl of PCR Master Mix, 2 µl of Primer Mix, 0.1 µl of Internal Amplification Control, 0.4 µl of DNA Taq Polymerase, 0.9 µl of DNase-free water, and 2 µl of DNA template. PCR amplification conditions were the following: 95ºC for 9 min; 45 cycles of 95ºC for 20 s, 61ºC for 25 s, and 72ºC for 30 s; and 72ºC for 8 min.

After PCR, hybridization and detection were performed in the FT-PRO flow-through system (Figure 11). In short, pre-hybridization (5 min), hybridization of the heat-denaturated PCR product (5 min), and post-hybridization washes were performed at 46ºC; first blocking step (5 min) and enzyme conjugation (5 min) were performed at 25ºC; finally, post-conjugation washes, second blocking step, colour development (5 min) and washes, and reaction stop were performed at 36ºC. It is of note that after each incubation, the liquid was drained by

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flow-through in the device. The results obtained were recorded and automatically interpreted by the DiagCor software (Figure 11), and were confirmed visually by the researcher.

Figure 11. Reverse hybridization array (A), FT-PRO flow-through hybridization system (B), and Capture-PRO image capture system (C).

(B. http://www.diagcor.com/en/mdx-products/detail/ft-pro-flow-through-hybridization-system) (C. http://www.diagcor.com/en/mdx-products/detail/capture-pro-image-capture-system)

4.3.2. Multiplex PCR

The multiplex PCR test evaluated (Anyplex II MTB/MDR/XDR, Seegene, Korea) was aimed to detect mutations in rpoB, katG, inhA promoter, gyrA, rrs, and eis promoter associated with resistance to RIF, INH, FLQ, and second-line injectable drugs (Article II).

The method is based on (dual priming oligonucleotide) DPO and tagging oligonucleotide cleavage and extension (TOCE) technologies. DPO [105] consists on amplification primers composed by two fragments separated by a polydeoxyinosine linker: the longer 5’ fragment allows stable priming, and the shorter 3’ fragment allows target-specific extension. This technology enable specific multiplex amplification with longer primers without increasing the annealing temperature. TOCE technology consists on detection of target sequences by the use of pitcher probes, which hybridize to the target templates, and the catcher probes, which are the reporter molecules. The pitcher is composed of the targeting portion, which is specific for the target template, and the tagging portion, which does not anneal with the target template.

Upon amplification of the template, the tagging portion is cleaved and released. The released tagging portion is complementary to the capturing portion of the catcher and serves as a primer for extension. Upon extension of the catcher, fluorescence signal is generated (Figure 12).

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Figure 12. Principle of the Anyplex II MTB/MDR/XDR assay, based on dual priming oligonucleotide (DPO) and tagging oligonucleotide cleavage and extension (TOCE) technologies.

(www.seegene.com/neo/en/introduction/core_toce.php)

The multiplex PCR assay Anyplex II MTB/MDR/XDR includes a pitcher probe with a specific targeting portion for each of the mutations targeted (Article II). However, all the pitcher probes that target resistance to the same drug share a tagging portion that is complementary to a catcher probe with a specific fluorophore: HEX fluorophore (6-carboxy-2,4,4,5,7,7-hexachlorofluorescein succinimidyl ester) for RIF and FLQ resistance, and Cal Red 610 fluorophore for INH and injectable drugs resistance. Therefore, two PCR reactions were performed: one reaction for detecting resistance to INH and RIF (MTB/MDR), and another reaction for FLQ and injectable drugs (MTB/XDR) (Article II).

Following the manufacturer’s instructions, PCR reactions were prepared with 5 µl 4X MTB/MDR or MTB/XDR oligo mix, 5 µl 4X Anyplex PCR Master Mix, 5 µl RNase-free water, and 5 µl of DNA template. PCR amplification conditions were the following: 95ºC for 15 min; 50 cycles of 95ºC for 30 s, 60ºC for 1 min, and 72ºC for 30 s; 55ºC for 30 s; and the melting curve detection step at 55-85ºC for 5s/0.5ºC. After amplification, the presence of a melting curve indicates resistance to the corresponding drug, whereas absence of a melting curve indicates susceptibility, and the Anyplex software yields a table reporting the results (Article II).

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4.3.3. Reverse hybridization line probe assay

The reverse hybridization LPA evaluated (AID TB resistance, AID Diagnostika, Germany) was aimed to detect mutations in rpoB, katG, inhA promoter, gyrA, embB, rrs, and rpsL associated with resistance to RIF, INH, FLQ, EMB, KAN, AMK, CAP, and STR (Article III). The method is based on PCR amplification and reverse hybridization on three different nitrocellulose strips.

The test was performed following the manufacturer’s instructions. Briefly, the PCR reaction was prepared with 15 µl of Primer Nucleotide Mix, 2.5 µl of 10X polymerase buffer, 1.5 µl of MgCl2, 0.2 µl of GoTaq® Polymerase (Promega, USA), and 5.8 µl of DNA template. PCR amplification conditions were the following: 95ºC for 5 min; 14 cycles of 95ºC for 30 s and 63ºC for 2 min; 26 cycles of 95ºC for 10 s, 55ºC for 30 s, and 72ºC for 30 s; and 72ºC for 8 min.

Hybridization and detection were performed with AutoLipa (Fujirebio, Belgium), an automated washing and shaking device. In short, hybridization of the denaturated PCR product (30 min), and post-hybridization washes (15 min) were performed at 47ºC, and rinse, enzyme conjugation (30 min), post-conjugation washes, colour development (15 min), and reaction stop were performed at room temperature. The results obtained were interpreted by the researcher (Article III). For those cases with unspecific background bands after the hybridization step, DNA was diluted 1:10 and the assay was repeated from the PCR step.

4.3.4. Pyrosequencing

This pyrosequencing assay was aimed to detect mutations in gyrA, rrs, eis promoter, and embB associated with resistance to FLQ, KAN, AMK, CAP, and EMB (Article IV).

Individual PCR reactions for each genomic region were prepared in a final volume of 25µl with 15 µl of ReadyMix™ Taq PCR Reaction Mix with MgCl2 (Sigma Aldrich, USA), 0.4 µM of each forward and reverse primer [65] (Article IV), 7.8 µl of DNase-free water, and 2 µl of DNA template. PCR amplification conditions were the following: 95ºC for 12 min; 45 cycles of 94ºC for 30s, 60ºC for 1 min, and 72ºC for 2 min; and 72ºC for 7 min.

After PCR, ssDNA was prepared by serial washings with a vacuum preparation tool and deposited in the pyrosequencing 96-well plate, where the pyrosequencing primer [65] (Article

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IV) was annealed. The pyrosequencing plate and the cartridge with the reagents (enzyme mix, substrate mix, and nucleotides) were loaded in the pyrosequencer, where the reaction took place (Figure 13). Pyrosequencing results were interpreted by analysing the pyrograms and the associated sequence.

Figure 13. Vacuum preparation tool (A) and pyrosequencing device (B).

(A. Sample Preparation Guidelines for PSQ™96 and PSQ 96MA Systems. Biotage)

(B. EpiTect™ and PyroMark™. A novel Relation setting Standards for the reliable Detection and accurate Quantification of DNA-Methylation. Qiagen)

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