A Rapid and Sensitive Chip-based Assay for Detection of rpob Gene Mutations Conferring Rifampicin Resistance in Mycobacterium tuberculosis (TB). Wanyuan Ao, Steve Aldous, Evelyn Woodruff, Brian Hicke, Larry Rea and Rob Jenison. Great Basin Corporation Salt Lake City, Utah Barry Kreiswirth Public Health Research Institute Tuberculosis Center University of Medicine and Dentistry of New Jersey Newark, New Jersey
TB and Drug Resistance A Challenge in the Developing World 8.8 million people infected with TB in 2010. 1.4 million died of TB in 2010. A leading killer of people with HIV infection. >95% of TB deaths occur in the developing countries. Due to limited resources and poor health care systems. Multi drug, extremely drug and totally drug resistant (MDR, XDR and TDR). Delayed identification and appropriate treatment. Poor sensitivity of diagnostics. A low cost and effective diagnostic assay is urgent.
rpob Gene and Drug Resistance The front drugs for Tb: rifampicin Rifampcin binds to RNA polymerase and interferes with RNA synthesis. Mutations in TB rpob gene, the beta subunit of RNA polymerase, have been reported to be correlated with TB drug resistance. The majority of the mutations (>95%) have been found to be within a 81 bp core region for the rpob gene. In addition, > 80% of the reported drug resistance cases have frequent mutations in 3 hot spots (codons: 516, 526 and 531 of rpob).
TB rpob Core Sequence and Clinically Relevant Mutations
Probes Covering the Core Sequence of the rpob Gene WT6.8.1 Amplification: Helicase Dependent Amplification (HDA) Isothermal, simple and low cost.
The Assay Process HDA Amplification and Chip Detection Amplicon Biotin HRP TMB Chip HDA Hybe Wash Wash Read Probe
Synthetic Reference Mutation Chip Data Wildtype No Template Controls MAP Examples of Some Mutations DC Detection Control HC1 Hybridization Control 510caT 513Gaa 516gGc 522tTg 526Tac 531Ccg
Performance of Probe Set Against Reference Mutations 100 10 1 Probe 1 Probe 2 Probe 3 Probe 4 Probe 5 Probe 7 Intensity WT128 508Gcc 509aCc 510caT 511cCg 511cGg 513cCa 513Gaa 513Aaa 513cTa 515Gtg 516gGc 516Tac 516gTc 518aTc 522tTg 522CAg 522tGg 526cCc 526Tac 526Gac 526caG 526Aac 526cGc 526cTc 529cAa 531tTg 531tGg 531Ccg 533cCg 533Gtg Mutation
Specificity with Synthetic Mutations Total 29 synthetic mutations were tested. 28/29 (>96%) were clearly identified as mutants. 1/29 (<5%) (codon 508Gcc) could not be discriminated with this probe set. The probe was poorly able to detect this mutation (Delta Tm = 0) Fortunately, this is a rare mutations, accounting <0.1% rifampicin resistance cases in TB.
Clinical Isolates Chip Data Wildtype No Template MAP Mutant Samples DC Detection Control HC1 Hybridization Control L511P 514 515 Insert H526D S531L D515G;L533P
100 10 Mutation Intensity (WT strain) (L511P) (L511P) (514 515 insert) (D516V) (H526C) (H526D) (H526D) (H526L) (H526R) (H526R) (H526Y) (H526Y) (H526Y) (S531L) (S531L) (S531L) (L533P) 1 Performance of Probe Set Against Clinical Isolates Probe 1 Probe 2 Probe 3 Probe 4 Probe 5 Probe 7
Specificity with Clinical Isolates Total 10 wildtype and 26 rifampicin resistant specimens were tested 10/10 (100%) wildtype specimens were identified correctly. 24/26 (92.3%) rifampicin resistant specimens were also classified correctly as mutants. The two mis classified specimens were confirmed to be a mixture of wildtype and mutant template population by sequencing. Cases like these could be a challenge.
Assay Limit of Detection 10 copies 3 copies 1 copy 0 copy Hybridization Control Probe 1.3 One single copy of genomic template per reaction Amplification time: 40 min.
Assay Specificity
Summary A Rapid and low cost TB assay has been developed. Wildtype TB and majority of clinically relevant drug resistance mutations could be detected. > 95 % of the drug resisitance Tb cases covered in the assay. Assay sensitivity: > 95% Assay specificity: All Tb complex strains as positive, no others will be detected. Limit of detection: one single copy in 40 minutes. A low cost automated instrument is being configurated.