The Place and Importance of Pcr-Rflp Method

Tuberculosis (TB) is one of the most ancient infectious diseases of human beings, and it is still in a leading position among infectious diseases as a cause of morbidity and mortality. In recently years, the HIV epidemics and the use of drugs that depress the immun system have resulted in an increase in infections [1,2]. Mycobacteria other than Mycobacterium tuberculosis (MOTT) infections are getting increase in human [3]. Key factors in the control of tuberculosis are rapid detection, adequate therapy, and contact tracing to arrest further transmission [4,5]. Determination of the species of mycobacteria, that are active agents in mycobacteria infections is important in developing appropriate treatment protocols and collecting epidemiological data. Conventional biochemical methods and phenotypic tests for species differentiation of Mycobacteria are laborious and time-consuming and frequently require specialized testing that is beyond the capacity of clinical laboratories. Various genotypic methods for the rapid identification of mycobacteria have been used in recent years [6-9]. With in the these methods, polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) method was described by Plikaytis et al. for differentiating


Introduction
Tuberculosis (TB) is one of the most ancient infectious diseases of human beings, and it is still in a leading position among infectious diseases as a cause of morbidity and mortality. In recently years, the HIV epidemics and the use of drugs that depress the immun system have resulted in an increase in infections [1,2]. Mycobacteria other than Mycobacterium tuberculosis (MOTT) infections are getting increase in human [3]. Key factors in the control of tuberculosis are rapid detection, adequate therapy, and contact tracing to arrest further transmission [4,5]. Determination of the species of mycobacteria, that are active agents in mycobacteria infections is important in developing appropriate treatment protocols and collecting epidemiological data. Conventional biochemical methods and phenotypic tests for species differentiation of Mycobacteria are laborious and time-consuming and frequently require specialized testing that is beyond the capacity of clinical laboratories. Various genotypic methods for the rapid identification of mycobacteria have been used in recent years [6][7][8][9]. With in the these methods, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was described by Plikaytis et al. for differentiating

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among slowly growing Mycobacterium species by PCR and restriction fragment length polymorphism analysis in 1992 [10]. This method is an easy, rapid and inexpensive method of identifying mycobacteria species in a single experiment when comprasion with the other molecular typing methods. This technique has been applied to several genes, for example 16S ribosomal DNA (rDNA), hsp65, dnaJ, groES and rpoB genes. The fundamental mechanism of this method is based on evaluation of the gene coding for the 65-kDa heat shock protein by PCR and restriction enzyme analysis. The PCR-RFLP was routinely used for the taxonomic separation of rapidly growing mycobacteria, identification of mycobacteria strains [11][12][13][14].
The success of this method, that yields results quite rapidly, varies according to standardization of the method used of each step, activity of restriction enzymes employed, markers of molecular weight, algorithm and experience of the user. In all these studies the algorithm describing the mycobacteria species is based on the use of two restriction enzymes (BstEII and HaeIII) and separation of the restriction fragments on an agarose gel. PRA patterns are then interpreted by converting the running distance in electrophoresis to apparent molecular size. Difficulties in PCR-RFLP interpretation may stem from similarities in a number of band sizes that are critical for discrimination of species and are not sufficiently resolved by agarose-based gel electrophoresis [15][16][17][18][19]. In this study, we were aim to evaluate the place PCR-RFLP method in routine use and its application practice by the hsp65 gene PCR amplification.

DNA extraction
A loopful of cells were suspended in 500µl of TE buffer (10mM Tris , 1mM EDTA , pH 8.0 ) in a 1.5ml screw-cap microcentrifuge tube. The suspension was centrifuged at 15,000xg for 10min. The supernatant was removed and then the pellet was washed twice with 500µl of TE buffer and then resuspended with 200µl of the same buffer. The samples were incubated in a boiling water bath for 20 min centrifuged, and supernatants containing DNA was transferred to clean microcentrifuge tubes and kept at -20°C until used [13].

Restriction digestion and analysis
All steps of the test were carried out in accordance with the recommendations of the manufacturer (Salubris Inc.,Turkey). For BstE II digestion, 20µl of PCR product was added directly to a mixture containing 0.5µl ( ~ 5U) of enzyme, 2.5µl of restriction buffer, and the mixture was incubated for 60min at 2h. Similarly, 20µl of product was incubated for 18h at 37°C in a solution containing Hae III enzyme, the corresponding buffer .

Evaluation of restriction patterns
After digestion, 4µl gel loading buffer (3% orange G, gliserol in water) was added ,and 10µl of the mixture was loaded onto a Nusieve 3:1 agarose gel (FMC Bioproducts) and 8% polyacriylamdie gel. Fragments were visualized by staining with ethidium bromide. Products were evaluated with a NuSieve 3:1 agarose gel and polyacrilamid gel electrophoresis (8%). In the stage of evaluation, Mayker B and Mayker H (Salubris Inc., Turkey) molecular weight standards specific to enzyme restriction regions Bst E II and Hae III, that contains certain band size were used. 21 standard strains of mycobacteria were also analysed with this method and M.tuberculosis H37Ra was used as a control in every run [13].

Results
In the beginning stage of our study ,all steps in house PCR-RFLP method were evaluated comparatively with standardized Mycobacteria typing kit . In standardization procedure using reference strains, same PCR products (441bp) were obtained

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with both methods (Figure 1) obtained amplification product was cut by two separate restriction enzymes in accordance with reference literature and protocols of manufacturer. Final product were evaluated with 8% polyacrilamid gel electrophoresis and 4% Nusieve Agarose (Figure 2). While Hae III enzyme yielded similar results with both methods , with Bst E II enzyme band patterns inconsistent with algorithm table and results of procedure same enzyme was evaluated in comparatively with new products and at different concentrations. In all studies ,similar results were obtained with the same enzyme. Therefore, study was continued with a different standardized enzyme.  A total of 80 mycobacterial isolates were evaluated by using routine biochemical tests and PCR -RFLP. All the strains identified as M.tuberculosis by PCR-RFLP were identified as the same by biochemical tests and colony morphology. The 21 standard mycobacterial strains used as controls reproducibly produced RFLP patterns in accordance with the patterns previously reported in other studies. The number and rates of species isolated are shown in Table III. Of 80 mycobacteria strains, 70(87.5%) were identified as Mycobacterium tuberculosis complex and 4(5%) as Mycobacterium scrofulaceum, 3(3.75%) as Mycobacterium gordonae. 3(3.75%) strains displaying different restriction patterns could not be identified as they were found in the algorithm reported in the literature. The resurgence of tuberculosis and apperance of multidrugresistant strains of M.tuberculosis have intensified the need for the increased use of rapid methods for isolation and identification of mycobacteria from patients. Mycobacterial species identification methods depending on colony morphology, pigment formation and biochemical analysis are very cumbersome and require several week [2,20,21]. Commercially available probe based methods are limited to identification of only limited number of species and expensive. Methods based on DNA sequencing are not practical enough to apply routinely and require expensive equipment. PCR-RFLP is simple to perform, easy to read, and reproducible and is used in several laboratories to identify MOTT. Mycobacterial identification by PCR-RFLP depends on amplification and restriction enzyme analysis of hsp 65 gene ,which exist in all mycobacterial species but show Advances in Biotechnology & Microbiology sequence differences from one to the other [10][11][12][13][14][15] (Figure 3). These differences are identified by restriction enzymes that recognize and cut spesific DNA sequences. Obtained fragments are separated by electrophoresis and the size of fragments are compared to molecular weight marker. The most important stage in PCR-RFLP method is the enzyme cut of amplification product. In cases when enzymatic activity is low, this procedure cannot be performed in the expected period and misleading results may be obtained during evaluation. The activity of the enzymes used in the test varies with storage conditions employment period, heat during reaction and production characteristics. Therefore, in order to minimise any mistakes during evaluation, material to be used in all steps of the test should be controlled according to certain standards prior to the test. In the initial step of our study, MYCO TYPE ALL® (Salubris inc) kit was used as the standard for optimisation of the test and control of restriction enzymes [16]. Band patterns obtained with both tests were evaluated comparatively in algorithma [13,15,17]. While there was consistency between two methods in all steps of the test, in the evaluation made in in-house PCR -RFLP metho after cut with BstE II enzyme, different band patterns were identified in algorithma. Hence, study was continued with another enzyme the activity of which was controlled with the same method. After the cut procedure with enzyme, gel with suitable characteristics and concentration for the seperation of fragments should be used. Two methods used for this purpose are polyacrilamid gel (8-10%) and electrophoresis 4% NuSieve agarose. Both methods have certain advantages and disadvantages. The preparation of the former method is more practical and makes gel manipulation easier, but can not differentiate bands at similar magnitude. On the other hand, lather method enables detailed differentiation at suitable concentration and defines bands lower than 100bp clearly. It is disadvantage is that its preparation and gel manipulation is difficult.

Discussion
Fragments in our study were evaluated with both methods. Although NuSieve agarose method was easier to employ, it was observed that it could not make differentiation as clear as polyacrylamide gel electrophoresis. In the evaluation stage of PCR-RFLP method, in order to differentiate species according to algorithm, the most important criteration is the selection of molecular weight markers containing suitable band size .The use of markers involving very far on very near band magnitude may cause incorrect evaluations. Therefore, the most commonly used molecular weight marker is In group and type differentiation, reference algorithm and Mycobacteria typing kit were evaluated comparatively with the modified algorithm. While similar resutls were obtained in group differentiation by both algorithms, in type differentiation algortihm produced by the manufacturer yielded more detailed information. Of 80 mycobacteria isolate investigated in our study, 70(87.5%) was identified as Mycobacterium complex, 4(5%) as Mycobacterium scrofulaceum and 3(3.75%) as Mycobacterium gordonae. 3 isolates with different band patterns could not be defined by either algorithm.

Conclusion
In conclusion, PCR-RFLP method is a rapid, practical and reliable method that can be used routinely in laboratories for typing mycobacterium strains isolated from clinical samples. Restriction enzymes and molecular weight standards play an important part in the efficacy of the test. It is suggested that quality control of materials used should be made comparatively with standards before the test and that more than one molecular weight standards at proper band size should be used in order to minimise mistakes at the stage of evaluation.