A modified low denaturing temperature PCR (LDT-PCR) method combined with DNA microarray technique is deve loped in our lab for quick and effective identification of various mutations in an 81 base pair region of Mycobacterium Tuberculosis (MTB) ribosome RNA polymerase subunit B (rpoB) gene associated with rifampin resistance. By incur poration of wild type (wt) allele fragments that had been PCR amplified previously, the target PCR fragments coming from mutant clinical MTB samples were codenaturized with incorporated wt type allele fragment at 94°C and then let them randomly form matched structures (homoduplex) and allele mismatch-containing structures (heteroduplex), re spectively, when the temperature cooled down to 70°C. After the temperature was raised to 80°C, the heteroduplex dou ble stranded fragments were preferentially denatured and resulted in PCR amplification as well as fluorescence incur poration. Since the homoduplex fragments need a higher temperature to be denatured, they were kept in dou ble-stranded status at that temperature and failed to be PCR amplified. By hybridization of LDT-PCR products with the probes spotted on microarray slides, the fluorescent signals representing the presence of gene mutations were detected. We have tested this method on 35 clinical MTB samples and obtained satisfied results.
Tuberculosis is the second leading cause of adult mortality among infectious diseases and is responsible for about 2 million deaths per year worldwide. The World Health Organization has estimated that in the next two decades 150 million people will develop tuberculosis and 36 million individuals will die from tuberculosis if control of this disease is not improved [
Many of the mutations that cause drug resistance in M. tuberculosis are point mutations in known chromosomal genes [
MTB rpoB is the only target of rifampin which is the most important anti-TB drug. A region containing 81 base pairs of nucleic acids in rpoB gene has been found to be a key position corresponding to rifampin treatment; any mutations happened in this region and changed amino acid sequences will cause the failure of rifampin treatment [
Recent years, a LDT-PCR method has been developed for selective identification of low-level unknown mutations [15-17].
In order to efficiently screen out drug-resistance MTB strains, our lab developed a modified LDT-PCR method combined with DNA microarray technique with a simple measurement to identify various nucleic acid mutations in MTB rpoB gene and obtained satisfactory results.
Strain H37Rv (No. 27294/TMC 102; ATCC*, Manassas, VA) served as the source of wild-type (i.e., natural sequence) M. tuberculosis genomic DNA. Clinical isolates of M. tuberculosis and the clinical drug resistance-susceptibility profiles of these isolates were obtained from collaborators at the National Institutes of Health (Bethesda, MD) and the Korean Institute of Tuberculosis, Korean National Tuberculosis Association (Seoul, South Korea). M. tuberculosis genomic DNAs were isolated (in a biosafety level 3 lab) from cultured bacteria using a standard phenol:chloroform method. Cultured M. tuberculosis was transferred to a 1.5 ml Eppendorf tube, pelleted by centrifugation and suspended in 0.36 ml of Marmur’s solution (0.1 M NaCl, 0.01 M EDTA, pH 8.0). Forty µl of 5% SDS, 2 µl of 20 mg/ml proteinase K, and 2 µl of 20 mg/ml RNase A were added to the suspension. The M. tuberculosis lysate was incubated at 37˚C overnight. An equal volume of buffersaturated phenol was added into the digest and thoroughly mixed by vortexing for 10 min. After centrifugation the top phase was transferred into a clean tube, extracted with an equal volume of chloroform:isoamyl alcohol (24:1) by vortexing for 10 min, and then centrifuged again. The top phase was transferred to a clean tube, and 0.1 volume of 3 M sodium acetate (pH 4.8) plus 2.5 volume of absolute ethanol were added. After vortexing briefly, the mixture was kept at −20˚C for 2 h and centrifuged at full speed for 15 min. The pellet was washed with 0.5 ml of 70% ethanol, dried, and suspended in 200 µl distilled water. The presence of genomic DNA was confirmed by agarose gel electrophoresis.
The design of PCR primers and probes used for microarray analysis was based on the published genome sequence of M. tuberculosis [
This experiment was composed of two steps of PCRs, or conventional PCR and LDT-PCR, both were performed in a GeneAmp® PCR Instrument System 9600 (Applied Biosystems, Foster City, CA).
For conventional PCR, the following protocol was used: Each 20 µl of PCR reagent contained 1× TaKaRa buffer, 200 µM each of dNTPs, 5 pM each of forward and reverses, 50 - 100 ng of M. tuberculosis DNA, and 1 units of TaKaRa Taq DNA polymerase. Cycling consisted of 94˚C for 3 min, followed by 35 cycles of 94˚C for 30 sec, 54˚C for 20 sec, and 72˚C for 45 sec. The final extension was 72˚C for 5 min. The size of the PCR products was analyzed by agarose gel electrophoresis. 5 µl of each PCR product was loaded in each lane.
For LDT-PCR, each 20 µl of PCR reagent contained 1× TaKaRa buffer, 5 pM each of forward and reverses, 1 µl of conventional PCR products from detected M. tuberculosis DNA sample and 1 µl of conventional PCR product from wild type H37Rv sample, 0.5 µM of Cy5 dye-labeled dCTP, and 1 units of TaKaRa Taq DNA polymerase. Cycling consisted of 94˚C for 2 min and then cooled down to 70˚C for 10 min, followed by 1 cycle of 80˚C for 30 sec, 54˚C for 20 sec, and 72˚C for 2 min. The LDT-PCR products were purified with ethanol precipitation method and finally eluted into 30 µl of distilled water.
For verification of the wild type and mutant clinical isolates of M. tuberculosis strains, all the PCR products from the samples used in this study were sequenced by the Core Lab in the Department of Health and Human Services, Center for Biologics Evaluation and Research Facility for Biotechnology Resources (U.S. Food and Drug Administration, Rockville, MD).
DNA oligonucleotide probes were diluted in spotting solution (50 M NaOH, 0.01% SDS) at a concentration of 100 µM and spotted onto CSS-silylated glass microscope slides (Telechem International Inc., Fisher Scientific, Suwanee, GA) with an OmniGrid AccentTM robotic microarrayer (Genomic Solutions, Ann Arbor, MI). The average spot size was 400 µm. Total 8 sub-arrays, each contained double spots of same rpoB probe, were spotted on each slide.
The probe slides were pre-treated with 0.2% SDS at room temperature overnight and washed with distilled water 3 times for 5 minutes followed by rinsed in isopropanol for 1 min and than air dried. For hybridization of dye-labeled DNA targets and oligonucleotide probes, an equal volume of purified targets was mixed with hybridization solution (50% formamide, 10× SSC, 0.2% SDS, 0.2 mg/ml sperm DNA). Three µl of mixed target solution was added to one of subarray probe areas on the slide, covered with a cover slip and hybridized at room temperture in a moisturized chamber for 4 - 6 hours. This was followed with a series of washing solutions that descended from 4× SSC to 0.5× SSC, each for 5 min at room temperature. The results of hybridization on slides were visualized using a GenePix 4000B Array Scanner (Axon Instruments, Inc., Union City, CA) at 635 nm (Cy5). The data were analyzed with GenePix Pro 4.0 software (Axon Instruments, Inc.). For the data figures presented in this paper, each pattern of red spots was reproduced from the scanner computer screen diagram. Each red spot represents a successful hybridization between a specific Cy5 fluorescent dye-labeled target DNA and oligonucleotide probe.
The designation of LDT-PCR was based on following idea: In a short DNA fragment (<200 bp), if there is a nucleic acid mismatched structure, the fragment will be denatured at a lower denaturing temperature as compared with non-mismatched fragment. Since the binding of primers to their positions on template and the extension of PCR can only be processed in a complete denaturing status of the template. Thus, a suitable lower denaturing temperature that should enable double-strands of mismatched template to be separated but without completely denaturing homological template will result in mutant structure amplification by PCR.
In our experiment, after being heated at 94˚C for 2 min, both allele rpoB PCR fragments, which had been amplified by previous conventional PCR from an unknown sample and a wild type (H37Rv) sample respectively and co-existed in a same PCR tube, would be completely denatured. After the temperature cooled down to 70˚C and left for 10 min, the denatured fragments would randomly re-natured to form four different combinations in a consistent ratio as about one-fourth of each combination. If there were a mutation in unknown sample fragments, the half the mutant fragments would form a heteroallele structure with wild type fragments at 70˚C (
copied by PCR extension and resulted in fluorescence incorporation (
in
Based on the sequencing results, 6 of the 35 samples we tested have mutations at position 531; 2 of them have mutations at position 526, and one has mutation at position 516. Rests of the samples are wild type with no mutation in the detecting area. With the method we developed, all 9 mutant samples showed positive signals as displayed in
A simple and reliable method for identification of various type nucleic acid mutations in a small area of DNA sequence has been developed. The method can be used to quickly screen out point mutation associated drug-resistant MTB strains from clinical samples. We believe thatthis detection method can be employed to identify various
genomic mutations in other applications. A suitable lower denaturing temperature for a specific target is critical in this method. The determination of this suitable temperature depends on the size and nucleic acid compositions of amplified PCR fragment.