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A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens
A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens
Journal of Microbiology and Biotechnology. 2016. Jan, 26(1): 180-189
Copyright © 2016, The Korean Society For Microbiology And Biotechnology
  • Received : January 07, 2015
  • Accepted : October 05, 2015
  • Published : January 28, 2016
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About the Authors
Wenfei Xue
State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China
Jingfu Peng
State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China
Xiaoli Yu
School of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University, Wuhan 430023, P.R. China
Shulin Zhang
Department of Medical Microbiology and Parasitology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China
Boping Zhou
The Third People's Hospital of Shenzhen, Shenzhen 518000, P.R. China
Danqing Jiang
State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China
Jianbo Chen
The Third People's Hospital of Shenzhen, Shenzhen 518000, P.R. China
Bingbing Ding
School of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University, Wuhan 430023, P.R. China
Bin Zhu
Baio Technology Limited Company, Shanghai 200233, P.R. China
zhubin@baio.com.cn
Yao Li
State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Science, Fudan University, Shanghai 200433, P.R. China
zhubin@baio.com.cn

Abstract
The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem-PCR) was applied to avoid interference between different primers in conventional multiplex-PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.
Keywords
Introduction
Mycobacterium tuberculosis (MTB) remains a serious threat to global human health [18 , 19] . The spread of multidrugresistant tuberculosis (MDR-TB) is resulting in high fatality rates [2 , 12 , 23] . MDR-TB comprises TB that is resistant to at least isoniazid (INH) and rifampicin (RIF).
Non-tuberculosis mycobacteria (NTM) may cause symptoms similar to MTB, while having different virulence attributes and drug resistance patterns compared with M. tuberculosis . The identification of MTB/NTM therefore forms a critical part of TB diagnosis and treatment.
Conventional drug susceptibility testing (DST) takes too much time. Other culture methods such as Bactec MGIT 960/BactTAlert [26] and phage technique [17 , 27] also have the limitation of time [29] .
The mechanisms of INH and RIF resistance were clarified in the last century [9 , 16 , 28] . These studies meant that rapid molecular detection methods could be developed for testing TB drug resistance.
Currently available molecular methods include real-time fluorescence-based quantitative PCR (such as GeneXpert), solid-phase hybridization assays, sequencing, electrophoresis-based techniques, denaturing high-performance liquid chromatography, multiplex-allele-specific PCR, and mismatch analysis [1] . These assays face different problems such as limited amount of covered loci, high costs, and sample preparation.
Most PCR-based assays use conventional multiplex PCR (m-PCR) in sample preparation, which is limited by the amplification conditions. Another problem is that the high concentration of primers present typically yields elevated background readings and reduces the amplification efficiency [3] . This limits the number of genes that can be tested at one time. Templex PCR (tem-PCR) is a platform that uses a target-enriched m-PCR method. It uses nested gene-specific primers at extremely low concentrations to enrich specific targets during the initial PCR cycles, and relies on universal forward and reverse primers at high but unequal concentrations to achieve exponential but asymmetric amplification [15] .
The enzymatic color microarray (ECM) technique is the combination of an enzyme-based color reaction and a microarray (biochip) technology [6] . Briefly, the ECM technique is carried out as follows. Primers with one terminus modified with biotin are used to amplify the target sequences and the resulting PCR products are therefore end labeled with biotin. Probes specific for the target loci are immobilized on the surface of a microarray according to the classical mode of modification with amidocyanogen. The amplified and biotin-labeled target sequences are then hybridized to the corresponding probes immobilized on the surface of the microarray. The microarray is then incubated with streptavidin-alkaline phosphatase conjugates, which undergo affinity binding to the hybridization products mediated by the biotin-streptavidin interaction. The chromogenic substrate of alkaline phosphatase, 5-bromo-4-chloro-3-indolyl phosphate p -toluidine salt, is used in conjunction with an oxidant, nitroblue tetrazolium, to produce a deep or lyons blue color.
In this study, we report an enzymatic color reaction-based biochip detection assay used no only for RIF and INH resistance of TB, but also for the identification of MTB/NTM. The assay includes an asymmetric m-PCR/tem PCRbased amplification method, biochip hybridization, and an enzymatic color reaction. Specific software is used for data acquisition and automated interpretation. The assay detects a variable region of 16S rDNA between MTB and NTM, codon 315 of the katG gene, a part of the regulatory region of the inhA gene, the 81 bp RRDR of the rpoB gene, and the most frequently observed mutations in these genes.
Materials and Methods
- Clinical Specimens and Genomic DNA Extraction
In total, 276 clinical specimens, including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 clinical culture isolate specimens, were tested ( Fig. 1 ). Of the 243 clinical culture isolates, 102 were from Wuhan Medical Treatment Center. The others were from the Third People’s Hospital of Shenzhen. Isolates from Wuhan were routinely cultured with egg-based Löwenstein-Jensen medium at 37oC. DST of these samples was done using the absolute concentration method according to “TB Diagnostic Laboratory Testing Procedures.” M. tuberculosis culture isolates previously identified by conventional biochemical methods were confirmed by 16S rRNA gene sequencing.
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Procedure of clinical specimen testing and analysis.
Isolates from Shenzhen were selected from the cultures grown in Bactec MGIT 960. MTB and NTM were discriminated by Bactec MGIT 960 too. DST of these samples was done using the method by the World Health Organization/International Union Against Tuberculosis and Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance.
Each of the other clinical specimens were from Shenzhen and divided into two portions. One portion was cultured using Bactec MGIT 960 and underwent DST analysis like isolates from Shenzhen. The other portion was treated to perform enzymatic color reaction-based biochip assay.
For culture isolate specimens, bacterial colonies were collected, resuspended in 200 μl of lysis buffer (10 mM Tris-HCl, pH 8.0,0.1 mM EDTA, 1.0% (v/v) Triton X-100) and mixed. The suspension was incubated at 80℃ for 2 h for decontamination, boiled for 10 min, and then centrifuged at 13,000 rpm for 15 min. The supernatant was stored at 20℃ until use.
For sputum specimens, alveolar lavage fluid specimens, and pleural effusions, 2-3 volumes of 4% NaOH was added to the samples and the mixture agitated at 140 rpm for 30 min at 37℃. Then 1 ml of completely liquefied sample mixture was added to a new centrifuge tube and centrifuged at 13,000 rpm for 10 min. After discarding the liquid, the sediment was mixed with 1 ml of normal saline and then centrifuged at 13,000 rpm for 10 min. The liquid was discarded and the sample was treated in the same way as for the culture isolate specimens.
- Primers and Probes
Primers were designed using Primer Premier 5/Oligo 6 software (Premier Biosoft, CA, USA). Strict NCBI BLAST verification was performed. The reverse primers for biochip hybridization were labeled with a biotin group at the 5’ ends. Oligonucleotide probes were labeled with an amine group at the 5’ ends. A poly(T) 10 chain was added to the 5’ ends to optimize the thermodynamic properties of the probes. Primers and probes were obtained from Life Technologies Co., Ltd. (Shanghai, China). The primers and probes used are shown in Table 1 .
Design of primers and oligonucleotide probes.
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Design of primers and oligonucleotide probes.
- Standard Clones
Standard clones for the sensitivity and specificity testing were established using E. coli DH5α strains and the pUC-T TA Ligation Kit (CoWin Bioscience Co., Ltd.). PCR was performed as follows: 95℃ for 10 min, 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 30 sec repeating for 30 cycles, and elongation at 72℃ for 10 min. The 25 μl PCR system contained 2.5 U of Taq DNA polymerase (CoWin Bioscience Co., Ltd.), 40 μM dNTP (CoWin Bioscience Co., Ltd.), and 0.2 μM forward and reverse primers. The standard clones included the wild-type rpoB , inhA , and katG genes, and the mutant versions of rpoB (CTG511CCG, GAC516GGC, CAC526AAC, and TCG531TTG), inhA (C-15T), and katG (AGC315ACC).
- Biochip Preparation
Probes were dissolved in TE buffer (pH 7.6), and then mixed with 2× spotting buffer. The probes were printed onto aldehyde-coated slides and covalently bound to the surface via the amine labels on their 5’ ends. Each probe was printed in three replicates with an Affymetrix 417 arrayer. For the loci of rpoB 511, 516, 526, 531, and 533, katG codon 315, and the 15 bp of the inhA regulatory region, negative control probes were printed in three replicates to set the cutoff value. A positive control probe (kit BST06013; BaiO Technology Co., Ltd.) was printed in three replicates in every line of the array to indicate a positive hybridization and color reaction. The biochips were incubated at room temperature for 2 h and stored at 4℃. Probes were printed as shown in Table 2 .
Probes printed in this study.
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Probes with names containing “NC” represent negative control probes. “P0” represent the 16S rDNA probe for MTB/NTM identification.
- M-PCR and Tem-PCR
The 25 μl m-PCR system contained 2.5 U of Taq DNA polymerase. The concentration of forward and reverse primers and dNTPs was 0.2 μM, 0.6 μM, and 40 μM, respectively. The concentration of template was adjusted according to experiment requirements. In clinical specimen testing, the template volume was 1 μl. Uracil-DNA glycosylase (1 U/μl; CoWin Bioscience Co., Ltd.) and dUTP (CoWin Bioscience Co., Ltd.) were used to prevent carryover contamination during amplification. The m-PCR assay was performed as follows: 50℃ for 2 min, 95℃ for 10 min, 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 30 sec, repeating for 30 cycles, and elongation at 72℃ for 10 min.
The 25 μl tem-PCR system contained 2.5 U of Taq DNA polymerase, 0.1 U of Uracil-DNA glycosylase, 0.2 μM forward super primer, 0.6 μM reverse super primer, 8 nM gene-specific primers, 40 μM dUTP, and 40 μM dNTP. The template concentration was set according to experiment requirements. In clinical specimen testing, 1 μl of template was used. Amplification was performed as follows: (i) 50℃ for 2 min, (ii) hot start (15 min at 95℃), (iii) enrichment stage (10 cycles consisting of 30 sec at 94℃, 30 sec at 55℃, and 1 min at 72℃), (iv) tagging stage (10 cycles consisting of 30 sec at 94℃ and 90 sec at 72℃), (v) amplification stage (35 cycles consisting of 30 sec at 94℃, 30 sec at 65℃, and 30 sec at 72℃), and (vi) extension stage (10 min at 72℃). All PCR products (3 μl) were analyzed by electrophoresis on 1.0% agarose.
- Biochip Reaction and Data Analysis
Biochip hybridization was performed in a BR-526 hybridization instrument (BaiO Technology Co., Ltd.), using a hybridization and enzymatic color reaction kit (BST06013, BaiO Technology Co., Ltd.) and 20 μl of PCR products. The hybridization temperature was 40℃. The hybridization program was set according to the protocol in the enzymatic color reaction kit [6] . The hybridization testing took 2.5 h. The slides were washed with ddH 2 O, dried, and then analyzed with a BE-2.0 scanner (BaiO Technology Co., Ltd.). Chromogenic signal intensities were quantified and exported using the Array Doctor 2.0 software (Baio Technology Co., Ltd.). The signal threshold for detecting probe spots for different loci was set at the mean ± 3 SD (standard deviation) of the respective negative control probe signals.
- Experiment Condition Optimization
Gradient experiments were conducted. According to the results, the annealing temperature for m-PCR was set as 55℃; the concentrations of reverse primer in m-PCR and the reverse universal primer in tem-PCR were 0.6 μM; the concentration of gene-specific primer was 8 nM; the probe concentration was 10 μM; the hybridization temperature was 40℃; and the hybridization time was 30 min.
- Sensitivity and Reproducibility
Target DNA sequences of nine standard clones were diluted to 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 100, 50, and 10 copies per PCR and used as sensitivity standards. Pure water (NCG1) and four smear and culture negative digested sputum samples (NCG2) was used as negative control samples. Both m-PCR and tem-PCR were used for sample preparation. To determine the reproducibility of the system, eight samples with 100 copies per PCR and eight samples with 10 5 copies per PCR were tested in six replicates as reproducibility standards, and samples of NCG1 and NCG2 were tested in six replicates. As mentioned in the Results section, tem-PCR needs a higher concentration of PCR template than m-PCR; thus, we applied m-PCR in clinical specimens biochip testing.
- DNA Sequencing and DST
PCR products were sequenced by Invitrogen. Drug susceptibility data of clinical specimens were collected from the Wuhan Medical Treatment Center and the Third People’s Hospital of Shenzhen. DST was performed with the BACTEC MGIT 960 system [8] at these hospitals.
Sequence information used in this study was from Mycobacterium tuberculosis H37Rv; the GenBank accession numbers are as follows: rpoB , 888164; katG , 885638; inhA , 886523.
The strain (ATCC No. 25618D) and genome DNA (ATCC No. 25618D-2) of Mycobacterium tuberculosis H37Rv are available at the American Type Culture Collection (ATCC).
No human and animal experiments were conducted in this study. No ethics approval was required.
Results
- Sensitivity and Reproducibility
Samples with a concentration of 50 copies per PCR could be successfully detected when using m-PCR for sample preparation ( Fig. 2 ), whereas the sensitivity was 500 copies per PCR when tem-PCR was used. All the negative control samples in reproducibility testing gave negative results, and all the results of the positive controls (including wild-type samples and mutant samples) corresponded to the results of DNA sequencing, indicating that the accuracy of the method was 100% ( p < 0.05).
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Results of the detection sensitivity experiment.

, , and show the results of sensitivity testing of wild-type probes used to detect wild-type samples. The template concentrations in the PCRs were 50, 500, and 5,000 copies per PCR in , , and , respectively. , , and show the results of sensitivity testing of the wild-type probe used to detect wild-type samples. The template concentrations in the PCRs were 50, 500, and 5,000 copies per PCR in , , and , respectively. The results showed that the sensitivity for and wild-type samples was 50 copies per PCR.

- Detection of Mixed DNA Samples
Three groups of mixed standard clone DNA samples of the rpoB , katG , and inhA genes were tested. Each group was a mixture of the wild-type and a mutant type DNA, with a concentration ratio gradient of 19:1, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 1:19. The template concentration was 10 3 copies per PCR. Allele sequences in mixed samples were successfully detected at the concentration ratio of 1:9 (10%), with a total concentration of 10 3 copies per PCR ( p < 0.05) ( Fig. 3 ). The single mixed-genotype sample present among the 294 clinical specimens was also tested and the result was consistent with that of DNA sequencing ( Fig. 4 ).
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Results of mixed sample testing.

Red boxes indicate the position of wild-type probes and yellow boxes indicate the position of mutant probes. ( and ) Testing results of mixed samples containing wild-type sequences and TCG531TTG mutant sequences. ( and ) Testing results of wild and mutant type mixed samples of the gene. ( and ) Testing results of wild and mutant type mixed samples of the gene. The samples in , , and contained wild-type DNA and mutant DNA at a ratio of 9:1, and the samples in , , and contained a ratio of 1:9. The concentration of the templates was 10 copies per PCR.

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Testing result of a clinical mixed specimen.

The figure indicates that the sample contains both wild-type DNA and GAC516GTC mutant DNA; the result was confirmed by DNA sequencing.

- Genotyping of Clinical Specimens
m-PCR was applied to biochip testing of the clinical specimens. Among the 276 specimens, 40 were NTM specimens and 236 were MTB specimens. According to DST data, 87 of the 236 MTB specimens were RIF-resistant and 78 were INH-resistant. The assay successfully distinguished all 40 NTM specimens from 236 MTB specimens, which means the sensitivity and specificity of MTB/NTM genotyping was 100%. The results of biochip assay, sequencing, and DST assay are listed in Table 3 . In the genomic region covered by the probes, results of biochip assay were in 100% agreement with DNA sequencing. Compared with DST assay, the results of the biochip assay showed concordance rates of 97.46% (230/236) for RIF resistance detection (83/87 for RIF-resistant specimens, with a sensitivity of 95.40%; 147/149 for RIF-susceptible specimens, with a specificity of 98.66%) and 96.19% (227/236) for INH resistance detection (73/78 for INH-resistant specimens, with a sensitivity of 93.59%; 154/158 for INH-susceptible specimens, with a specificity of 97.47%) ( Fig. 5 ). In detection of mutant specimens, the assay discovered 95.79% (91/95) of rpoB , 95.38% (62/65) of katG , and 76.92% (10/13) of inhA mutant specimens.
Comparison of the results of DST assay and biochip detection of RIF and INH susceptibility in 236M. tuberculosisclinical specimens.
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Comparison of the results of DST assay and biochip detection of RIF and INH susceptibility in 236 M. tuberculosis clinical specimens.
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Results of clinical specimen detection.

() The test result of a wild-type clinical specimen, which did not have any mutations in the regions covered by the probes used. The system interprets this result as an RIF- and INH-susceptible MTB sample. () This image indicates that the sample DNA has mutations of CAC → GAC in codon 526 of the gene and AGC → ACC in codon 315 of the gene. The system interprets this result as an RIF- and INH-resistant MTB sample. () This image indicates that the sample DNA did not match the 16S rDNA probe, thereby indicating that it was a NTM sample. The red boxes show the key probe positions.

Discussion
The emergence of MDR-TB is a major global health problem. Real-time PCR assays are widely used in TB testing, including the TaqMan probes [11] , molecular beacons [30] , and bioprobes [10] . Real-time PCR-based assays have the advantages of speed (testing time of 1.5–2 h) and avoiding cross-contamination. However, the amount of genes tested in a reaction is limited. Sophisticated instrumentation with high associated costs [25] is required. It is also difficult to distinguish allele sequences in mixed samples.
Solid-phase hybridization assays [1] , including linear probe assays and DNA microarrays (DNA biochips), are commercially available. The GenoType MTBDR and MTBDRplus assay shows a sensitivity of 98.1% and specificity of 98.7% for RIF resistance testing and a sensitivity of 84.3% and specificity of 99.5% for INH [21] . Another assay, LiPA, shows a sensitivity of 82-100% for RIF resistance, and a specificity of 92-100% [22] . However, the amount of probes printed on a single strip limits the application of linear probe assays, and thus the testing results could be incomplete and rough [4 , 24 , 31] . Meanwhile, linear probe assays also have a lower level of automation of operating and data analysis.
DNA microarray assays [7] have been used in the testing of rifampicin, pyrazinamide, streptomycin, isoniazid, and ethambutol [5 , 13 , 14] . Fluorescence-based microarray assays require expensive equipment and reagents, which could limit their application. More genes were proved to be related with drug resistance in recent years [20] , while m-PCR limits the number of genes tested in a single reaction.
Our enzymatic color reaction-based biochip assay has several advantages compared with other probe-based hybridization assays in the field of testing flux, result completeness, versatility, sample preparation, and testing time. The color reaction biochip improves the flux of a single reaction and amount of genes tested. The assay covers MTB/NTM identification and RIF and INH drug susceptibility testing, and contains probes for the most frequently observed mutation types. More genes and loci can be included in a single testing reaction. The assay directly shows the mutation type of 104 of the 130 rpoB mutant specimens, 74 of the 94 katG mutant specimens, and 16 of the 20 inhA mutant specimens, and thus provides detailed and reliable data for clinical diagnosis. The assay was optimized for detection of mixed samples and can specifically detect allele sequences in mixed samples at aconcentration proportion of 10% of the total. The sensitivity was 50 copies per PCR, thus reducing the sample requirements. The detection system realized a high level of automation, as the monitoring and measurement of the hybridization and color reaction operate automatically, and specific software is used in data processing and reporting. Application of the tem-PCR technique avoids the interference of primers that occurs in conventional m-PCR. In this study, various clinical specimens, including sputum specimens, alveolar lavage fluid specimens, pleural effusion, and clinical culture isolate specimens, were tested. The system shows high sensitivity and specificity ( Table 3 ), and the whole testing progress can be finished in 4.5 h.
The color-reaction-based biochip in this study provides a potential assay for MTB/NTM genotyping and detection of TB drug resistance. This assay is rapid and semi-automatic, and could be a new tool for the clinical diagnosis and treatment of TB.
Acknowledgements
This work was supported by a grant from the National Key Technologies R & D Program of China during the Twelfth Five-Year Plan Period (No. 2013ZX10003003-004-002), Shanghai Science and Technology Commission (Nos. 12XD1422000 and 13DZ2252000), and Grant D20121809 from the Educational Commission of Hubei China.
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