Journal of Microbiological Methods 70 (2007) 301 – 305 A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis Xiaodong Cheng a,1, Jianfang Zhang a,b,1, Liu Yang, Xiuli Xu a, Jianyun Liu a, Wenbin Yu a, a Department of Clinical Laboratory, Xijing Hospital, the Fourth Military Medical University, Xi'an, China b Department of Microbiology, the Fourth Military Medical University, Xi'an, China Received 26 September 2006; received in revised form 27 April 2007; accepted 1 May 2007 Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in asingle assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patternswere observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using theculture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determinedto be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.
2007 Published by Elsevier B.V.
Keywords: Multidrug resistant; Tuberculosis; Multiplex PCR; Single-Strand Conformational Polymorphism Analysis (SSCP) and transmission of resistant cases. Currently, the detection ofdrug resistance in Mycobacterium tuberculosis is primarily Tuberculosis (TB) has a long and continuing history of based on phenotypic drug susceptibility testing, which involves causing worldwide morbidity and mortality. The emergence of time consuming culture of the slow growing M. tuberculosis multidrug-resistant TB (MDR-TB), defined as resistance to at least isoniazid (INH) and rifampin (RIF), the 2 principal first- line anti-TB drugs, poses an important threat to TB control.
understanding of the genetic mechanisms of M. tuberculosis MDR-TB reduces responses to standard short-course chemo- drug resistance, and the advancement of molecular technologies therapy with first-line anti-TB drugs, leads to higher mortality in recent years, have allowed the development of more rapid and treatment failure rates, and increases the period of molecular methods to detect mutations in genes implicated in Most of the established methods for detecting drug resistance of MDR-TB occurred worldwide in 2000, accounting for 3.2% are based on single gene or single drug testing. However, it is clear of all new TB cases (). Prompt detection of anti- that the simultaneous detection of genetic mutations implicated TB drug resistance is essential for controlling the development in both INH and RIF resistance would provide a more accurate and spread of MDR-TB, as it facilitates appropriate and timely assessment of MDR-TB. The reverse line blot assay described by delivery of anti-TB therapy, reducing overall cost of treatment is the first reported attempt to combinedifferent targets in a single assay for prediction of multiple anti-TB drug resistance. Following Mokrousov's report, ⁎ Corresponding author. Tel.: +86 29 84775455; fax: +86 29 82527499.
described the use of a molecular beacon to simultaneous- 1 Both authors contributed equally to this work.
ly detect INH and RIF resistance-associated mutations in 0167-7012/$ - see front matter 2007 Published by Elsevier B.V.
X. Cheng et al. / Journal of Microbiological Methods 70 (2007) 301–305 RIF. All of the 12B medium vials used in the test were pre-tested on a BACTEC 460TB instrument to establish a CO2atmosphere in the vial and to screen out any vials with a GI(Growth Index) of 20 or more. Bacterial suspension (0.1 mlactively growing M. tuberculosis cultures in 12B medium, GI500-799) was used to inoculate each of the BACTEC 12Bmedium vials containing the drugs. For the control vial, a 1:100dilution, made by transferring 0.1 ml of the suspension into9.9 ml of Diluting Fluid (Becton Dickinson and Company,Sparks, MD) was used. After mixing thoroughly by inverting at Fig. 1. Multi-PCR-SSCP electrophoresis gel fragment patterns. Note: The lane least 10 times, 0.1 ml of this dilution was used to inoculate the MR, MI and MK represents rpoB, inhA and katG gene of H37Rv, respectively.
MR has 3 ssDNA bands and the reason may be that there are different alleles. MI control 12B medium vial (without a drug). The vials were has 1 ssDNA band and it may because the 2 ssDNA has affinis three- incubated at 37 ± 1 °C and tested daily at approximately the dimensional conformation. Lane M represents multi-PCR-SSCP of H37Rv.
same time each day (± 2 h) on a BACTEC 460TB. When the Lanes 1–7 represent multi-PCR-SSCP of clinical isolates. Lines 1 and 4 have no control vials reached a GI of 30 or more, the results were mutations; lines 2 and 5 have katG mutation; line 3 has rpoB and katG interpreted as follows: If the ΔGI was less in the drug vial than mutations; line 6 has inhA and katG mutations; line 7 has all three genesmutations.
the control, the population was deemed susceptible, if more, itwas considered resistant.
MDR-TB was defined as resistance to at least INH and RIF.
M. tuberculosis complex, from cultures and smear positive sputa.
To ensure the reliability of the phenotypic susceptibility testing In addition, reported direct detection of results, we performed each phenotypic drug susceptibility multiple RIF and INH resistance mutations in M. tuberculosis testing twice at different times, using the same test system.
respiratory samples using real-time PCR. established a multiplex allele-specific PCR (MAS-PCR) method that simultaneously detected INH, RIF, and ethambutol (EMB)resistance-associated genetic mutations. Here we report the M. tuberculosis genomic DNA was isolated from each development of a Multi-PCR-SSCP (Single-Strand Conforma- culture using a standard phenol:chloroform method. Cultured tional Polymorphism Analysis) method that simultaneously M. tuberculosis was transferred to a 1.5 ml Eppendorf tube, detects INH and RIF resistance-associated genes katG, inhA, pelleted by centrifugation (12,000×g, 5 min) and suspended in and rpoB in a single assay, at a lower cost, and using less 0.36 ml of Marmur's solution (0.1 M NaCl, 0.01 M EDTA, pH demanding techniques and more economical equipment than the 8.0). SDS (40 μl of 5% solution), 2 μl proteinase K (20 mg/ml), and 2 μl RNase A (20 mg/ml) were added to the suspension.
The M. tuberculosis lysate was incubated at 37 °C overnight.
An equal volume of buffer-saturated phenol was added to thedigest and thoroughly mixed by vortexing for 10 min. The top phase was transferred to a clean tube after centrifugation(12,000×g, 5 min) and extracted with an equal volume of For this study we used a convenience sample of 116 chloroform:isoamyl alcohol (24:1) by vortexing for 10 min, and M. tuberculosis clinical isolates obtained from Xi'an Chest centrifuging (12,000×g, 5 min). The top phase was transferred Hospital. Each isolate represented a different patient. The to a clean tube, and 0.1 volume of 3 M sodium acetate (pH 4.8) identification of M. tuberculosis complex strains was based on plus 2.5 volumes of absolute ethanol were added. After conventional methods, including the niacin production test and vortexing briefly, the mixture was chilled at −20 °C for 2 h the nitrate reduction test, etc. As the purpose of the study was to and centrifuged at 12,000×g for 15 min. The pellet was washed develop a method for rapid detection of drug resistance- with 0.5 ml of 70% ethanol, dried, and suspended in 200 μl associated mutations of M. tuberculosis isolates, rather than to distilled water. The presence of genomic DNA was confirmed describe the prevalence of genetic mutations, all the available resistant isolates and pan-sensitive isolates were included.
Strain H37Rv (ATCC27294) served as the source of wild-type PCR primers for amplifying M. tuberculosis drug resistance genes A proportion method based on the BACTEC 460 radiometric system (Becton Dickinson and Company, Sparks, MD) was used for drug susceptibility testing. Aliquots of 0.1 ml drug solution were added to 4 ml BACTEC 12B medium vials and 2307 5′-CAGACG TTG ATC AAC ATC CG-3′ 305 prepared for final concentrations of 0.1 μg/ml INH and 2 μg/ml X. Cheng et al. / Journal of Microbiological Methods 70 (2007) 301–305 2.4. Establishment of the multi-PCR technique testing as the reference standard, we assessed the sensitivity andspecificity of the Multi-PCR-SSCP for detection of INH and RIF The design of DNA oligonucleotide primers used for PCR resistance. Sensitivity is defined as the probability of Multi-PCR- amplification was based on the published genome sequence of SSCP testing positive for drug resistance-associated mutations if the resistance was found by culture. Specificity is defined as the mutations in M. tuberculosis genes that are associated with drug probability of Multi-PCR-SSCP testing negative if the resistance was not found by culture. The reproducibility of the Multi-PCR- The amount of each primer pair used in this assay was first SSCP was examined by repeating the assay for all the study optimized to achieve maximum amplification in separate reactions isolates. The sequence results for M. tuberculosis H37Rv were using the same PCR program and reaction conditions. Next, the used as the reference for sequence comparison.
amount of each primer pair in the Multi-PCR was balanced toachieve acceptable amplification of all target genes. For each Multi-PCR reaction, a standard 25 μl reaction mixture was used.
Each reaction mix included 6 primers: 25 pmol in 1 μl, respectively.
The other reagents included in each reaction mix were 2 μl of200 μmol/l deoxyribonucleotide mix, 2.5 μl of Mg2+-free 10× PCR Based on the phenotypic drug susceptibility testing results, reaction buffer, 2.5 μmol/l MgCl2 1 μl, 1 μl Taq Polymerase Mix, 70 of the 116 isolates were identified as INH resistant, including 5 μl DNA solution containing 50 ng DNA template, and 7.5 μl 62 MDR-INH resistant and 8 non-MDR-INH resistant isolates.
PCR-grade water. The thermocycling parameters included an ini- Of the 116 isolates tested, 66 were identified as RIF resistant, tial denaturing at 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 50 °C including 62 MDR-RIF resistant and 4 non-MDR-RIF resistant.
for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 7 min.
Forty-two isolates were pansensitive.
The PCR products were examined for banding patterns by 2.0%agarose gel electrophoresis in 1× TAE buffer.
2.5. Single-Strand Conformational Polymorphism Analysis The three genes, katG, inhA and rpoB, in the 116 clinical isolates and H37Rv strain were amplified successfully in single The amplified DNA was mixed with an equal volume of formamide loading dye (97% formamide, 20 mmol/l EDTA and 0.05% bromphenol blue), denatured at 95 °C for 5 min, chilled on ice for 1 min and loaded onto 16 cm × 18 cm, 10% acrylamide:bisacrylamide (49:1) gels containing 0.2% glycerol.
Electrophoresis was performed using Protean II xi cells (Bio- Rad Laboratories), at 400 V for 5 min, then 200 V 16–20 h below 15 °C in 1× TBE buffer. The gels were silver-stained used to provide reference wild type patterns on each gel.
2.6. Cloning of PCR amplimers and DNA sequencing Amplimers were cloned into a PGEM T-Easy vector (Promega, Madison, WI), and transformed into competent Escherichia coli cells, following the protocol recommended by the manufacturer. For each transformation, 3 clones were selected and incubated overnight in LB broth at 37 °C in a shaking rotary incubator (225 rpm). Plasmids from the selected clones were extracted using a QIAprep Spin Miniprep kit (Qiagen), and identified by EcoRI digestion, then sequenced in both directions using M13 forward and reverse primers and an Applied Biosystems DNA sequencer (Model 3700 or 3730).
2.7. Evaluation of the Multi-PCR-SSCP assay Correlations between the DNA sequences and Multi-PCR- SSCP results were determined to evaluate the usefulness of Multi- PCR-SSCP in determining genetic mutations in clinical isolates.
Using the results of culture-based phenotypic drug susceptibility X. Cheng et al. / Journal of Microbiological Methods 70 (2007) 301–305 PCR reactions, except in the case of 4 katG deletion mutants.
specificities of the Multi-PCR-SSCP and DNA sequencing Compared to strain H37Rv, 46 isolates had katG gene methods for detecting INH and RIF resistance, and MDR, were mutations, 14 had inhA mutations and 58 had rpoB mutations.
assessed, using culture results as a reference. The specificities Thirty-eight isolates had simultaneous katG and rpoB mutations and sensitivities of the Multi-PCR-SSCP determined for each and 4 isolates had both inhA and rpoB mutations. Four isolates testing group were similar to the corresponding specificities and had inhA and katG mutations and 2 isolates had mutations in all Of the 70 isolates that were INH-resistant by culture, 56 were identified as INH-resistant by Multi-PCR-SSCP, resulting in a detection sensitivity of 80%. Four of the 8 non-MDR INH-resistant isolates showed mutant patterns, 4 had wild type Partial katG gene sequencing revealed mutations in 48/70 patterns. The specificity of Multi-PCR-SSCP for INH-resis- (68.6%) INH-resistant isolates, including 44/62 (71.0%) MDR tance detection was 100%, as 46 isolates that were susceptible isolates and 4/8 (50%) non-MDR-INH-resistant isolates. The 4 to INH by culture all showed the expected wild-type SSCP MDR-INH-resistant isolates had complete katG deletions, based on the reproducible absence of PCR product. The other For RIF resistance detection, the sensitivity of the Multi- 44 isolates possessed 46 point mutations, 4 base insertions and 4 PCR-SSCP assay was 81.8% with the identification of 54 of the deletion mutations Ten isolates had two mutations.
66 culture-proven RIF-resistant isolates. Of the 50 culture- Sequencing of the partial inhA gene revealed 13 point proven RIF-susceptible isolates, 4 had the SSCP pattern of a mutations and 1 single base deletion within 8 codons in 24 resistant strain, producing a specificity of 92% for RIF de- isolates (), accounting for 24/70 (34.3%) of the total tection. Mutations at codons 516 and 531 of the rpoB gene were INH-resistant isolates. Of the 22 INH-resistant isolates with no found in the 4 isolates by DNA sequencing. However, results of mutation in the sequenced region of the katG gene, 12 isolates repeated phenotypic drug susceptibility testing still classified had mutations in the inhA gene. Of the 10 isolates with no these 4 isolates as RIF-susceptible.
mutations in both genes, 6 were MDR isolates and 4 were non- Of the 62 culture-proven MDR isolates, 44 were identified as MDR INH-resistant isolates. That means 56/62 (90.3%) of the MDR by Multi-PCR-SSCP and 46 by DNA sequencing. All of MDR isolates and 4/8 (50%) of the non-MDR INH-resistant the 54 non-MDR isolates defined by culture were classified as isolates had mutations in the katG and/or inhA genes ().
non-MDR isolates by Multi-PCR-SSCP. The sensitivity of the Sequencing of the 304-bp central region of the rpoB gene Multi-PCR-SSCP method for MDR detection was 71.0%. Spe- revealed point mutations at 8 different codons in 60 isolates.
Most mutations were found at codons 531, 526, and 516. Inaddition, 2 point mutations were detected at codon 528. Four isolates were found to have dual mutations at 2 different codons,either codons 516 and 526, or 531 and 528. Two non-MDR Our objective was to develop a molecular assay that allows INH-resistant isolates and 2 pansensitive isolates had mutations simultaneous detection of M. tuberculosis resistance to multiple at codons 526 (). Of the 66 RIF-resistant isolates, 10 anti-TB drugs, INH and RIF, the 2 determining drugs for MDR- MDR isolates had no mutations at this region of the rpoB gene.
TB. We developed a Multi-PCR-SSCP that targets the 3 genefragments in which INH, and RIF resistance-associated muta- tions are most frequently observed. Detection sensitivity and thespecificity of the new method were assessed in comparison with Culture-based phenotypic drug susceptibility testing is cur- the culture-based phenotypic method, using 116 clinical isolates rently used as the standard. Therefore the sensitivities and of M. tuberculosis. The results of this study indicated that thisnewly developed method has potential to identify more than80% RIF or INH resistant TB isolates in 24–48 h.
The reverse line blot assay reported by Mokrousov et al., Sensitivity and specificity of Multi-PCR-SSCP assay and DNA sequencing fordetecting M. tuberculosis resistance to INH, RIF among 116 clinical isolates which was the first attempt to detect multiple drugs in a singleassay, but it only included known hot-spot region mutations.
Compared to the reverse line blot assay, the MAS-PCR assay detection of INH resistance-associated mutations in settings where the prevalence of the commonly seen mutations is high among the resistant clinical isolates. However, it only targeted 6 reported the use of molecular beacons for simultaneous detection of INH and RIF resistance-associated mutations in M. tuberculosis from cultures and smear-positive sputa. This assay targets the same genomic loci as the MAS- PCR. The molecular beacons assay is clearly a useful method X. Cheng et al. / Journal of Microbiological Methods 70 (2007) 301–305 for the rapid detection of mutations implicated in INH and RIF resistance if resources are available. Compared with molecularbeacon-based detection, the MAS-PCR method is almost equal- Bassam, B.J., Caetano-Anolles, G., Gresshoff, P.M., 1991. Fast and sensitive ly efficient in terms of detecting the commonly seen genetic silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196 (1),80–83 Jul.
mutations implicated in INH and RIF resistance. However, the Canetti, G., Fox, W., Khomenko, A., Mahler, H.T., Menon, N.K., Mitchison, MAS-PCR can not include all mutations in the gene. Multi- D.A., et al., 1969. Advances in techniques of testing mycobacterial drug PCR-SSCP is not based on pre-defined or known mutations, so sensitivity and the use of sensitivity tests in tuberculosis control programs.
it may improve the sensitivity of detection. Multi-PCR-SSCP Bull. World Health Organ., Suppl. 41, 21–43.
combines 3 tests into a single assay, thereby substantially in- Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., et al., 1998. Deciphering the biology of Mycobacterium tuberculosis from the creasing the efficiency, and significantly reducing the cost, of complete genome sequence. Nature 396 (6707), 190 Nov 12.
detection. In addition, Multi-PCR-SSCP is less technically de- Dye, C., Espinal, M.A., Watt, C.J., Mbiaga, C., Williams, B.G., 2002. Worldwide manding and requires less expensive equipment, making it more incidence of multidrug-resistant tuberculosis. J. Infect. Dis. 185, 1197–1202.
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Laszlo, A., Rahman, M., Raviglione, M., Bustreo, F., 1997. Quality assurance A major limitation to molecular genetic detection of drug program for drug susceptibility testing of Mycobacterium tuberculosis in theWHO/IUATLD Supranational Laboratory Network: first round of profi- resistance by any technique is that such tests generally only detect ciency testing. Int. J. Tuberc. Lung Dis. 1, 231–238.
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important complementary method for TB drug susceptibility Mokrousov, I., Bhanu, N.V., Suffys, P.N., Kadival, G.V., Yap, S.F., Cho, S.N., et al., 2004. Multicenter evaluation of reverse line blot assay for detection of Another limitation of this test, particular to the current study, is drug resistance in Mycobacterium tuberculosis clinical isolates. J. Microbiol.
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Simultaneous detection of isoniazid, rifampin, and ethambutol resistance ofMycobacterium tuberculosis by a single multiplex allele-specific polymer- We thank Dr. Duan Yan of Xi'an Chest Hospital for collecting ase chain reaction (PCR) assay. Diagn. Microbiol. Infect. Dis. 53, 201–208.
bacteria strains. We thank all the staff of the clinical microbiologiclaboratory for providing assistance in the experiment.


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