Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase

Abstract

Background: Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research.

Methods: We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method.

Results: Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%).

Conclusions: We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.

Figure 1. Schematic representation of PCR amplification and sequences of primers for PCR and sequencing.

(A) Top: Site-directed mutagenesis using oligonucleotide is shown using K103N as an example. Desired mutations in the reverse transcriptase gene were engineered in two PCR fragments, then incorporated into a larger fragment (1050 bp, HXB2 location 2388–3425) by the second PCR, and cloned into pBluescript II SK (+) at XhoI-BamHI sites. Bottom: Negative strand cDNA was synthesized from patients’ plasma. After the first strand PCR using RT1F and RT1R as primers, Fragment a or Fragment c were amplified by nested PCR. (B) Primer sequences for PCR amplification and sequencing.

Figure 2. Validation of PCR-SSOP-Luminex assay using plasmids as templates.

(A) Agarose gel electrophoresis of amplified fragments. Lanes 1–6: Fragment a (547 bp). Lanes 7–14: Fragment c (512 bp). 1, wild type; 2, M41L-TTG; 3, M41L-CTG; 4, K65R-AGA; 5, 70R-AGA; 6, 70R-AGG; 7, wild type; 8, 103N-AAC; 9, K103N-AAT; 10, M184V-GTG; 11, T215Y-TAC; 12, T215F-TTC. (B) Median fluorescence intensities (MFIs). The plasmid in the test sample is indicated on the top of each panel. Oligoprobes used for detection are indicated at the bottom. Matched results are shown as black bars, mismatched results as white bars. Assays were performed in triplicate. The mean ± standard deviation is shown at the top of each bar.

Figure 3. Assay sensitivity in a mixture.

(A) Agarose gel electrophoresis of mixtures of amplified fragments. Left panel: Fragment a (547 bp). Right panel: Fragment c (512 bp). Wild-type:mutant ratio; Lanes 1 (10∶0); 2 (9∶1); 3 (8∶2); 4 (7∶3); 5 (5∶5); 6 (3∶7); 7 (2∶8); 8 (1∶9); 9 (0∶10). (B) Signals from wild type probes (black bars) and mutant probes (white bars) in each mixture. “% of signal” was calculated by “MFI of wild type or mutant signal” divided by “MFI of wild type plus mutant signal” and multiplied by 100. Triplicate experiments were performed three times. “% of signal” is shown with standard deviations.

Figure 4. PCR-SSOP-Luminex DR assay of clinical samples.

Results of 18 probes for 6 DR loci and 5 standard probes are shown. Each dot represents the mean of triplicates. Dashed line indicates the cut-off value, the lowest MFI among 5 standard probes (MFI = 837.5).

Figure 5. Improvement of the detection by the additional probes at K65 locus.

The ordinate shows the percentage of the genotype at K65 locus determined by the PCR-SSOP-Luminex DR assay. One hundred % is the sample number (74) successfully amplified by PCR.