Application Of The Luminex XTAG In Rapid Detection Of Multidrug Resistance In Mycobacterium Tuberculosis

ObjectiveMDR-TB is defined as resistant to at least rifampicin (RIF) and isoniazid (INH).Diagnosis of MDR-TB is of great value in the tuberculosis control and prevention.Conventional drug susceptibility testing (DST) using culture techniques will take2-3months for a conclusive result. Such a delay allows transmission of resistant strainsand at the same time the incorrect treatment will lead to aggravation of TB infectionin individuals. At present, a few assays are used in rapid MDR-TB diagnosis.Nonetheless, due to low accuracy, complicated manipulation, high cost or some otherproblems, these assays cannot be widely used in low-income and middle-incomecountries. It is very important and urgent to develop a rapid, inexpensive and simplemethod for these countries. In this study we reported an assay based on LuminexxTAG technology to detect MDR-TB.MethodsDrug resistance in M. tuberculosis usually arises by single point mutations inantibiotic target genes. In particular, RIF resistance is due mainly to mutations in an81-bp RIF resistance-determing region of the rpoB gene, and INH resistance isfrequently associated with mutations in the katG and inhA genes. The most frequentmutations in MDR-TB are as follows:①katG315(AGC→ACC, AGC→AAC),(INHresistance, mutation frequency is50%-90%)②inhA-15(C→T)(INH resistance,mutation frequency is6%-30%);③rpoB531(TCG→TTG, TCG→TGG), rpoB526(CAC→GAC, CAC→TAC)(RIF resistance, mutation frequencies are75%and10%,respectively).Firstly,8double-stranded DNA were synthesized which contained the target detectionsites and target alleles. Following PCR and purification, ASPE reactions were carriedout by different PCR products and allele-specific primers. The parameters of reactionwere optimized. Next,6PCR primers were designed to perform multiplex PCR with genomic DNA ofM. tuberculosis as template. Following the steps above, the final products weredetected by Luminex xTAG flow cytometer.Finally, we used the Luminex xTAG method to detect mutations in MDR-TB relatedgene codons in153M. tuberculosis clinical isolates. Meanwhile, the mutations werealso detected by a DNA-sequencing-based method to evaluate the Luminex xTAGmethod.ResultsThe results suggested that Luminex xTAG technology showed good feasibility andspecificity in detecting mutations in different target site, in alleles in same site and inmixed infection when synthesized DNA or genomic DNA performed as templates.The best annealing temperature of ASPE reaction was59℃. The best hybridizationtemperature was37℃. The best hybridization time was30min.The results of single genotype infection detected by Luminex xTAG technologyperfectly matched with those detected by sequencing methods. There were9isolatesdetected by Luminex xTAG method showed mixed infection, and7of them had thesame results with DNA sequencing method. While the other2isolates showed singlegenotype infection by DNA sequencing method. Maybe it was due to the limitation ofthe numbers of the plasmid purified for sequencing.Luminex xTAG was a rapid method with high accuracy and specificity for detectionof MDR-TB. It could detect MDR-TB in many isolates within one working day andperformed extremely well in mixed infection detection.ConclusionThe new method is very useful for rapid detection of MDR-TB clinical isolates inlow-income and middle-income countries.