A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay

Ye-Cheng Zhou, MS; Shu-Mei He, MS; Zi-Lu Wen, MS; Jun-Wei Zhao, PhD; Yan-Zheng Song, PhD; Ying Zhang, PhD; Shu-Lin Zhang, PhD

Disclosures

Lab Med. 2020;51(6):606-613. 

In This Article

Materials and Methods

M. tuberculosis Isolates and Drug Susceptibility Testing

All M. tuberculosis isolates examined in this study were from Wuhan Medical Center in China, according to the principles recommended by the World Health Organization (WHO) guidelines and our previous study.[22,23] A total of 54 isolates of M. tuberculosis consisting of 32 different types of resistant isolates and 22 fully susceptible isolates were tested. Drug susceptibility testing of original isolates to first-line drugs INH (0.2 μg mL−1), RIF (40 μg mL−1), streptomycin (STR; 4 μg mL−1) and ethambutol (EMB; 2 μg mL−1) was performed with the proportion method.[24] Analytical data concerning the drug-susceptibility tests of RIF and INH on the 54 isolates are shown in Table 1. Resistance to any of the 4 drugs was defined as greater than 1% growth in drug-containing medium, compared with growth in a drug-free control medium. Isolates resistant to at least INH and RIF were considered to be MDR-TB.

Oligonuleotide Primers and Capture Probes

The primers and probes were synthesized by Invitrogen (Thermo Fisher Scientific Inc.). Three pairs of primers, used for PCR amplification of rpoB, katG, and inhA regulatory regions, were designed using Primer Premier, version 5.0 software (PREMIER Biosoft) based on the M. tuberculosis sequence from GenBank. All 3 reverse primers were labeled with digoxigenin at the 5′ end to facilitate detection of the PCR product (Table 2). In addition, a total of 12 biotin-labeled probes to determine the resistance to RIF or INH in the PCR-microplate hybridization assay were designed, using Primer Premier, version 5.0 and OLIGO, version 6.0 (Molecular Biology Insights, Inc.) software. These probes were between 17 and 22 nucleotide long and carried a biotin at their 5′ ends.

All the capture probes comprised of 7 wild-type probes and 5 mutant probes specifically targeted sites where many mutations conferring drug resistance occurred: for rpoB, the 81-bp RIF RRDR; for katG, the-315 site (INH resistance); and for the inhA regulatory region, the -15 site (INH resistance). For each probe, the position of the potential mismatching in similar sequences was located in the center of the probe (Table 3). Finally, the specificity of PCR primers and specific probes designed was analyzed via a search of the GenBank database using the BLAST program.

DNA Preparation and PCR Amplification

Genomic DNA of clinical isolates was extracted, as previously described.[25] The 3 pairs of labeled primer (Table 2) were used for PCR. PCR amplification was performed in a 50-μL reaction volume containing 2 μL of template DNA, 1 × PCR buffer (10 mM Tris–HCl [pH 8.3], 50 mM KCl, and 3 mM MgCl2), 0.4 μM of each primer, molecular biology–grade water, 200 μM of dNTP, and 2.5 U of Taq DNA polymerase was synthesized by Invitrogen (Thermo Fisher Scientific Inc.). The reaction was performed in a EDC-810 model thermal cycler (Eastwin Life Sciences, Inc.).

Thermal cycling parameters were as follows: 94°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds; 58°C for 30 seconds; 72°C for 30 seconds; and a terminal extension step of 72°C for 10 minutes. PCR products were analyzed by electrophoresis on 1% agarose gels after ethidium bromide staining.

Detection of PCR Products by Specific Hybridization and ELISA

Detection of digoxigenin (DIG)–labeled amplified products was accomplished using a PCR-microplate hybridization assay. The 96-well microplates were coated with 100 μL of streptavidin (20 μg/mL in 0.05 M carbonate-bicarbonate buffer [pH 9.5]) at 4°C overnight and then rinsed 3 times with tribuffered saline (TBST; 150 mM NaCl; 100 mM Tris-HCl [pH 7.5]; 0.1% Tween-20 [v(volume)/v]). Unsaturated binding sites were incubated with 200 μL of blocking buffer (40 mM Tris-HCl [pH 7.4], 1% bovine serum albumin [BSA; w(weight)/v]) for 1 hour at 37°C, followed by 3 washings with TBST.

To demonstrate the feasibility of the hybridization and ELISA, all the tests were performed in duplicate. In detail, 5 μL of DIG-labeled PCR product was denatured with 15 μL of denaturation solution (100 mM NaOH; 0.1% Tween-20 [v/v]) at room temperature for 10 minutes. We mixed 225 μL of hybridization solution (300 mM NaCl; 100 mM Tris-HCl [pH 6.5], 10 mM ethylenediaminetetraacetic acid [EDTA], 0.1% Tween-20 [v/v]), containing 8 pmol per mL biotinylated type-specific individual probe, with the denatured DIG-labeled PCR product; the mixture was prewarmed for 10 minutes at 63°C. Aliquots (200 μL) were then immediately transferred to the wells of a streptavidin-coated microplate and incubated at 63°C in a water bath for 60 minutes. For removing the extra PCR-probe complexes, the wells were washed 4 times with TBST buffer. Then, the hybrids immobilized to the well were labeled by incubating 200 μL of antidigoxigenin antibody conjugated horseradish peroxidase, diluted 1:5000 in TBST for 30 minutes at 37°C. After 4 washes with TBST buffer, 100 μL per well of TMB enzyme substrate was added, and the reaction was carried out at room temperature for as long as 10 minutes. Absorbance at 450 nm was measured with a Microplate Reader (Agilent Technologies) after the reaction was stopped by the addition of 100 μL of 2 M H2SO4 to each well. Because we discovered that not all probes worked with equal efficiency, the criteria that gave a distinct discrimination between the various wild-type and mutant genotype needed to be applied for the interpretation of results acquired via PCR-microplate hybridization assay. Individual probes, except for RW2 and RM2 probes that gave OD values of greater than 1.0, were considered to have positive results, whereas those with values of less than 0.35 were considered to have negative results. Conversely, RW2 and RM2 probes that yielded an optical density (OD) of greater than 2.0 were considered to have positive results, and those with values of less than 1.0 were considered to have negative results. Results for a given specimen were considered valid only if hybridization pattern satisfied the aforementioned criteria.

DNA Sequencing

PCR products were purified by use of a E.Z.N.A Gel Extraction Kit (Omega Biotek, Inc.). Purified PCR products of 54 isolates from rpoB, katG, and the inhA regulatory region were sequenced (Table 1) by Shanghai Majorbio (Shanghai, China) on an ABI 3730XL automated sequencer (Thermo Fisher Scientific Inc.). The sequence data were assembled and analyzed by using Bioedit software, version 7.0.8. Also, VectorNTI software, version 11.0 (Thermo Fisher Scientific Inc), was used to compare the data with those in the H37Rv genome database (GenBank accession no. L27898).

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