| In 1993,the World Health Organization(WHO)took an unprecedented step and declared tuberculosis a global emergency.Multi-drug resistant tuberculosis(MDR-TB)is becoming a serious public-health concern in developing countries.The WHO has estimated that 9.6 million people fell ill with TB and 1.5 million died from the disease in 2014,an estimated 1 million children became ill with TB and 140 000 children died of TB.Globally in 2014,an estimated 480 000 people developed multidrug-resistant TB(MDR-TB).TB incidence has fallen by an average of 1.5% per year since 2000 and is now 18% lower than the level of 2000.China is currently ranked 1st among the 27 countries with the highest burden of MDR-TB and extensively drug-resistant tuberculosis(XDR-TB)in the world.Early diagnosis of drug resistance and DST-guided individualized chemotherapy are crucial for the optimal management of this disease.However,conventional DST of Mycobacterium tuberculosis still relies on culture of the bacilli and requires at least several weeks.Therefore,ineffective anti-tuberculosis regimens may cause acquired drug resistance and local recurrence during this period.Moreover,DST for tuberculosis spondylitis is not performed routinely in most resource-poor hospitals in China because of the biosafety concerns and inadequate infrastructure,which present a major hindrance for the treatment of the disease.Therefore,there is an urgent demand to develop rapid,accurate and feasible techniques for tuberculosis diagnosis as a complement to the pathology,and meanwhile to detect drug-resistant forms of spinal tuberculosis.In recent years,a multitude of molecular DST kits,such as INNO-Li PA,Genotype MDR-TBplus,DNA microarrays and Xpert MTB/RIF,have been developed for the detection of mutations associated with resistance to RMP and INH.Among the above molecular diagnostic technologies,the DNA microarray is based on the hybridization of DNA to oligonucleotide chips and is alr eady being applied to gene expression profiling,detection and genotyping of microorganisms,detection of mutation,and gene discovery.Gene chip is a kind of second-generation sequencing technology.As integrated analytical equipment,it had been extensively applied to biochemistry and medical diagnosis field because it can form thousands of functionalized probes to implement parallel analysis of target sample accurately and rapidly with high throughput.To the mycobacterium tuberculosis in different drug resistance degrees caused by numerous genetic locus mutations,the fast drug sensitivity detection by biochip technology contains advantages of technological feasibility and high throughput.The tuberculosis drug resistance detection by gene chip can be directly completed in the laboratory of comprehensive hospital;meanwhile,with the constant development and mature of gene chip technology,reduction of manufacturing as well as detection costs,it has been applied extensively.―Jingxin? Drug Resistance Detection Kit for Mycobacterium Tuberculosis‖,Chinese first tuberculosis fast drug resistance detection product researched and developed by Biochip Beijing Engineering Research Center can detect the drug resistance of two kinds of first-line drugs simultaneously such as rifampicin and isoniazid with high sensitivity and specificity,which accumulated valuable experience in this aspect.Therefore,this project group cooperates with Biochip Beijing Engineering Research Center to research and develop second generation of drug resistance detection chip for mycobacterium tuberculosis,which can analyze 8 genes,22 mutation sites and 38 mutants simultaneously,and detect the drug resistance situation of8 kinds of first and second-line anti-tuberculosis drugs,including RMP,INH,SM,EMB,LVFX,AMK and CPM.Objective:Our plans were to research and develop second generation of detection chip system for tuberculosis drug resistance based on ARMS-PCR-magnetic bead TagArray platform developed by Biochip Beijing Engineering Research Center so as to detect plasmids in different template concentrations and understand the sensitivity and specificity;then use second generation of chip to implement detection analysis on osteoarticular tuberculosis clinical isolates and evaluate its specificity,sensitivity,accuracy and feasibility.Methods:1.Obtained 24 strains of sequenced mycobacterium tuberculosis in different mutants from Beijing Center for Disease Control and Prevention and then extracted plasmids(each sample of bacterial strain only contain one kind of mutant).Implemented concentration detection after obtaining plasmids;diluted different plasmids into 1×103 copy/μl,1×103 copy/μl,1×104 copy/μl,1×105 copy/μl,1×106 copy/μl,and then carried out multiple PCR amplification parallel chip hybridization to analyze the sensitivity and specificity of this chip detection system to sample detection,and evaluate its feasibility as well as application prospect in practical clinical detection.2.Obtained 187 strains of clinical isolates extracted from patients with osteoarticular tuberculosis from Chongqing Infectious Disease Medical Center,among which 50 strains were sensitive bacteria and 137 strains were drug resistant bacteria in various types(all include drug sensitivity and sequencing result).Designed,optimized and prepared chip detection system,including primer and label screening,construction and optimization of multiple PCR amplification system,and optimization of hybridization system(this part was completed by Biochip Beijing Engineering Research Center).Then,used second generation of chip to implement drug sensitivity detection on the clinical isolates of 187 strains of mycobacterium tuberculosis,analyze according to sequencing result and phenotype drug sensitivity result,and discuss about the specificity,sensitivity,accuracy and feasibility of second generation of chip on detecting drug resistance of mycobacterium tuberculosis on first and second-line anti-tuberculosis drugs.Results:1.When detecting the 24 bacterial strains in different mutants with gene chip,they demonstrated the mutants in accordance with sequencing result in the sets with 1 ×103copy/μl of template concentration and above;while 29 sets showed false negative result in the sets with 1×102copy/μl of template concentration.2.Among the 126 rifampicin phenotype drug resistance strains,the chip detected 119 strains appeared mutation in rpo B gene and rpoB gene mutation was not detected in the other 7 strains;in the rifampicin phenotype sensitivity strains,the drug resistance locus of 9 strains of rpo B gene showed mutation were detected.When comparing with results of phenotype drug sensitivity,the sensitivity of chip detection was 94.40%,and specificity was 86.76%.Among the 118 isoniazid phenotype drug resistance strains,the chip detected 109 strains appeared mutation in relevant drug resistance gene locus and the mutation of drug resistance gene locus was not detected in the other 9 strains;when comparing with results of phenotype drug sensitivity,the sensitivity of chip detection was 92.37%,and specificity was 81.16%.Among the 126 ethambutol phenotype drug resistance strains,the chip detected 27 strains appeared mutation in locus emb B306,and the mutation in this drug resistance gene locus was not detected in the other 17 strains;in the ethambutol phenotype sensitivity strains,the mutation in locus embB306 was detected in 6 strains.When comparing with results of phenotype drug sensitivity,the sensitivity of chip detecti on was 61.36%,and specificity was 95.71%.Among the 102 levofloxacin phenotype drug resistance strains,the chip detected 81 strains appeared mutation in gyr A gene,and the mutation in this drug resistance gene locus was not detected in the other 21 strai ns;2 of the sensitivity strains were detected mutation in gyrA gene.When comparing with results of phenotype drug sensitivity,the sensitivity of chip detection was 79.41%,and specificity was 97.65%.Among the 112 streptomycin phenotype drug resistance strains,the chip detected 101 strains appeared mutation in relevant drug resistance gene locus and the mutation of drug resistance gene locus was not detected in the other 11 strains;when comparing with results of phenotype drug sensitivity,the sensitivity of chip detection was 90.17%,and specificity was 82.67%.Since amikacin,capreomycin and kanamycin(Second-line injectable drugs,SLID)are in cross drug resistance,the results were counted together(in clinical isolates,on bacterial strain will be considered as drug resistance strain as long as tolerating one or several of the drugs).Among the 67 phenotype drug resistance strains,the chip detected 52 strains appeared mutation in relevant drug resistance gene locus and the mutation of drug resistance gene locus was not detected in the other 15 strains;in the phenotype sensitivity strains,rrs1401 gene mutation was not detected i n 11 strains;its sensitivity was 79.10% and specificity was 90.83%.Conclusions:1.The gene chip can implement fast and accurate drug resistance detection when template concentration is not lower than 1×103copy/μl.Most samples in clinic can meet the detection condition while few samples may need 2-4 weeks‘ mycobacterium tuberculosis culture before detection.2.This research evaluated the accuracy and feasibility of using Tag chip magnetic bead method to implement drug resistance detection on mycobacterium tuberculosis clinical isolates RFP/INH/ EMB/ SM/SLID(AMK,CPM and KM)/FQS,among which RFP,INH,FQS and SLID(AMK,CPM,KM)showed high sensitivity and specificity that were close to phenotype drug resistance result.However,it is available to add eis promoter region detection to SLID(AMK,CPM,KM)to enhance sensitivity and bring higher detection accuracy.Molecular diagnosis showed that the drug resistance sensitivity of EMB was so low that it is likely to cause missed diagnosis of EMB resistanc e,but it still plays certain reference value to the formulation of individualized scheme in early chemotherapy due to its high specificity;especially the positive result that needs strong vigilance.This new Tag Array is a rapid approach of good biosafety and high sensitivity and specificity in resistance test of first and second line MTB drug for the isolated Mycobacterium tuberculosis strains,and is satisfying in clinical practice for rapid diagnosis of drug-resistant Mycobacterium tuberculosis strains. |
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