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Srinagarind Med J 2007: 22 (suppl) Detection and Differentiation of Mycobacterium Species in Clinical Specimens by Real-Time Polymerase Chain Reaction Sunisa Siriudom1, Kunyaluk Chaicumpar2, Viraphong Lulitanond2 and Wises Namwat2 1 Graduate student in Microbiology department Department of Microbiology, Faculty of Medicine, Khon Kaen University , Khon Kaen 2 PROCEEDING Introduction Tuberculosis is still a serious infectious disease in human. It is estimated that are 8 million new cases and 2 million deaths per year, worldwide1. The World Health Organization (WHO) is predicted incidence of tuberculosis are 12 million cases2. In Thailand, tuberculosis is the second of top ten mortality rate of infectious disease rank from Pneumonia (http:// epide.moph.go.th). The increase in mycobacterial diseases has stimulated the development of rapid efficient methods of diagnosis. Rapid diagnosis and species differentiation of the mycobacteria responsible for disease are important to infection control and antimicrobial therapy. For these reasons, rapid, simple, sensitive and specific methods for detection and differentiation of mycobacteria in clinical specimens were developed. In this era, nucleic acid amplification techniques have been accessible to the clinical mycobacteriology laboratory. The majorities of technologies are based on the polymerase chain reaction (PCR) and have been developed in house. 208 23 2550 PCR-based assays for the detection of M . tuberculosis approach the sensitivity and specificity of conventional culture but have the added advantage of being rapid. However, these procedures have problems including false-negative and false-positive. In addition, multiple steps are required in the amplification and detection steps involving user manipulations at each point of the assay that have the error and sample contamination. Real-time PCR techniques, involving fluorescent dye or fluorophores with spectrofluorometric have been used to develop a number of rapid and sensitivity assays for identification of many microorganisms. Fluorogenic probes, including molecular beacons and paired hybridization probes can be designed to specifically recognize a specific sequence from target gene and can enhance specificity and sensitivity over conventional PCR techniques. A real-time PCR technique for detection of mycobacteria has also been developed2- 5. It is able to detect a specific sequence during amplification and needs no hybridization or further processing time for analysis. Srinagarind Med J 2007: 22 (suppl) In this study, were designed primers and probes to develop and evaluate a rapid diagnostic test, real-time polymerase chain reaction for detection and differentiation of Mycobacterium species directly from clinical specimens. By the side of primers and probes target is 16S-23S rRNA internal transcribed spacer (ITS) region. Materials and Methods Primers and hybridization probes design for PCR In this study, primers and probes were designed by LightCycler probe design software 2.0 based on the multialignment analysis data. Primers were designed from the highly conserved region on the basis of 3' end of 16S rRNA and ITS sequences of mycobacteria, respectively. Whereas, the probes were choose base on varies polymorphic of ITS region sequences. The quality of primers and probes was analyzed for specificity with a BLAST search and BioEdit, for melting temperature by Oligocalc and for dimmers formation by Oligoanalyzer. Amplification oligonucleotide and realtime PCR Amplification oligonucleotides with Rank 9 primers were have amplicons size approximately a 214 bp fragment of ITS sequence from mycobacteria. These primers were included in a Results and discussion Bioinformatics analysis and primers design The ITS sequences has five conserved regions an several polymorphic region in the group of M. tuberculosis complex and M. avium complex. The bases sequence of each region that are conserve have 43 bp, 109 bp, 15 bp, 26 bp and 21 bp, respectively. The forward of primer Rank9 designed by select at the region 2 while the reverse of these primer was located at between region 4 and 5. By the way, the specific probe of each group was located among region 3 and 4. 23 2550 209 PROCEEDING Bioinformatics analysis The mycobacterial 16S-23S ITS region sequences were obtained from GenBank (http:// www.ncbi.nlm.nih.gov). Conservative and polymorphic of the ITS sequences of mycobacteria were sought in a multialignment with BioEdit software, CLUSTAL-W alignment. real time PCR reaction mix utilizing the QuantiMix EASY SYG KIT (Biotools, Spain) to evaluate their ability to amplify the ITS fragment from all of the mycobacterial species tested. The PCR reactions were amplified and analyzed using the Roche LightCycler software 2.0. The PCR program setting were 1 cycle of denaturation at 95 C for 10 min and 40 cycles of amplification, each cycle was 10 sec at 95 C , 10 sec at 60 C and 10 sec at 72 C with single read. Melting curve profiles were generated for each amplified product using the LightCycler over a temperature range of 55 C to 95 C at 0.1 C per second with continuous read. Construction of the Mycobacterial positive controls Using DNA isolated from an M. tuberculosis H37RV and M. avium. The TB ITS was amplified with set of primers MTB and M. avium were used setoff MACnew primers. The fragment was inserted into the pGEM cloning plasmid using the pGEM T-Easy Vector (Promega, USA). The construction of plasmid was confirmed by sequencing. PROCEEDING Srinagarind Med J 2007: 22 (suppl) Detection of mycobacteria with realtime PCR and conventional PCR Real-time PCR experiment using ITS primers with Sybr Green dye were experiment using ITS primers, Rank 9, with Sybr Green dye was completed successfully amplified all extracted DNA of mycobacteria. The amplification fragments were detected based on the increase of fluorescent signal that emitted when dye bind with the minor groove of double stranded oligonocleotide (Fig. 1). In this experiment, found that the negative control can be detected the fluorescence signal at lately cycle. Following the PCR phase, melting curve profiles were determined for each amplified fragment (Fig. 2). The melting point profiles of mycobacteria have only peak of melting temperature. Thus, a hybridization probe is needed for species differentiation. To this step, Rank 9 primer can be used to amplify the ITS sequence of mycobacterium species. The fluorescence signal that can detect from negative control is possibly caused of by primer dimmers formation. To verify the size of PCR products of real-time PCR, the 1.5 % agarose gel electophoresis were performed (Fig.3A). The result showed corresponding length of the amplicon, 214 bp. In the other hand, these primers were used to amplify mycobacteria by conventional PCR. There were two amplicons, the one specific bold band is true target (214 bp.) and another one is nonspecific band (~300 bp.) (Fig.3B). Why the same sets of primer are getting the difference result when use in conventional and real-time PCR?. The answer of question is these primers were designed from software that appropriated for real-time PCR but not for conventional PCR. Fig.1. Real-time PCR amplification of ITS fragment from 8 specie of mycobacterium. The amplification primers were designed to amplify the ITS from all species of Mycobacterium. PCR amplification of the ITS fragment was detected using the LightCycler 2.0 and Sybr Green I dye. 210 23 2550 Srinagarind Med J 2007: 22 (suppl) (A) PROCEEDING Fig. 2. Melting-curve profile for ITS fragments amplified from each Mycobacterium species. These melting points are difficult to distinguish to specie and not enough for identified M. tuberculosis complex from NTM. The hybridization probe is need for further improvement. (B) Fig. 3. Agarose gel electophoresis of amplified products from constructed positive control of real-time PCR with Rank 9 primer (A) and performed with conventional PCR (B). Panel A : Lane M = 100 bp., Ladder, Lane 1 = Negative control, Lane 2 = p-MTB-ITS and Lane 3 = P-MAC-ITS Panel B : Lane M = 100 bp., Ladder, Lane 1 = Negative control, Lane 2 = p-MTB-ITS, Lane 3 = P-MAC-ITS, Lane 4 = M. tuberculosis DNA and Lane 5 = M. avium DNA Conclusion The Rank 9 primers were designed by LightCycler 2.0 software for amplify ITS region of 16S-23S rDNA of mycobacterium species. Using Sybr Green dye for signal detection, it was successfully amplified all extracted DNA of both M. tuberculosis complex, M. avium complex and another specie is belong to genus Mycobacterium.. Its amplicon can be visualized by gel electrophoresis with the corresponding length, 214 bp. When using 23 2550 211 Srinagarind Med J 2007: 22 (suppl) PROCEEDING this primer in real-time PCR found one product that have size of amplicon about 214 bp. It can be amplify of both M. tuberculosis complex, M. avium complex and another specie is belong to genus Mycobacterium. This pair of primers was also used for performing with conventional PCR , found the nonspecific of product about 300 bp, were found. The result suggested that concluded that this primer is not appropriate for real-time conventional PCR. Hybridization probe for strain differentiation and verification of the protocol in clinical specimen is needed for complete the protocol. Acknowledgment This work was supported by Faculty of Medicine Research Grant, Khon Kaen University. Keywords: R eal-Time Polymerase Chain Reaction, Internal transcribed spacer region (ITS), Mycobacterium tuberculosis complex, Mycobacterium avium complex 212 23 2550 References 1. Huggett JF, McHugh TD, Zumla A. Tuberculosis: amplification-based clinical diagnostic techniques. Int J Biochem Cell Biol 2003;35:1407-12. 2. Kraus G, Cleary T, Miller N, Seivright R, Young AK, Spruill G, et al. Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR. Mol Cell Probes 2001;15:375-83. 3. Lachnik J, Ackermann B, Bohrssen A, Maass S, Diephaus C, Puncken A, et al. Rapid-cycle PCR and fluorimetry for detection of mycobacteria. J Clin Microbiol 2002;40:3364-73. 4. Shrestha NK, Tuohy MJ, Hall GS, Reischl U, Gordon SM, Procop GW. Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR. J Clin Microbiol 2003;41:5121-6. 5. Miller N, Cleary T, Kraus G, Young AK, Spruill G, Hnatyszyn HJ. Rapid and specific detection of Mycobacterium tuberculosis from acid-fast bacillus smear-positive respiratory specimens and BacT/ ALERT MP culture bottles by using fluorogenic probes and real-time PCR. J Clin Microbiol 2002; 40:4143-7.

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