Abstract:
The present invention relates to a kit and a method to identify  M. tuberculosis , which may include an approach relating to isolating chromosomal DNA from a biological sample, such as a culture, conducting a real time PCR to obtain an amplicon, digesting said amplicon separately with restriction enzymes NruI and BamHI to obtain a digestion product separating said digestion product into its constituents in a gel; and analyzing the restriction enzyme digestion pattern.

Description:
CROSS-REFERENCE REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of priority to Indian Patent Application Number 911/DEL/2014, filed on Mar. 29, 2014, the contents of which is hereby incorporated by reference herein 
       SEQUENCE LISTING 
       [0002]    The present application is being filed along with a sequence listing in electronic format. The sequence listing is provided as a file entitled SeqList_ANAND013 — 001AUS.txt, created Mar. 27, 2015 which is 1 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. 
       FIELD OF THE INVENTION 
       [0003]    The present invention relates to a method to identify  M. tuberculosis  using the restriction enzymes NruI and BamHI. 
       BACKGROUND OF THE INVENTION 
       [0004]    Tuberculosis is a major cause of death due to a single infectious agent. Early diagnosis and treatment of tuberculosis not only improve patient outcome but also helps to control this disease by reducing transmission. Unfortunately, conventional methods of bacteriological diagnosis of tuberculosis are time-consuming and labor-intensive. Moreover, although  Mycobacterium tuberculosis  is the most important  Mycobacterium  species from a public health perspective. Nontuberculous mycobacteria (NTM) are increasingly being reported in various parts of the world. Though NTM are ubiquitous organisms, normally found in the environment, several species are pathogenic to humans. Hence, rapid identification of  Mycobacterium  spp. is imperative for appropriate diagnosis and treatment, especially in immuno-compromised individuals. 
         [0005]    The limitations of conventional methods of diagnosis of tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive and specific. However, their expense and the need for technical expertise make them difficult to be used in regions endemic for tuberculosis. 
         [0006]    PRA techniques have been developed for several mycobacterial genes, such as hsp65, the 16S-23S rDNA spacer and rpoB. However, most of these techniques require the use of an algorithm to identify the  Mycobacterium  species. Also, the formation of short restricts or small differences between bands needs the use of Nusieve or Metaphor agarose which are expensive; or polyacrylamide gel, which is difficult to handle, thus making the assay technically demanding. 
         [0007]    Accordingly, to overcome the problems encountered in the prior art, the inventors of the present invention provide a method to identify  M. tuberculosis  using the restriction enzymes NruI and BamHI, as described herein below. 
       SUMMARY OF THE INVENTION 
       [0008]    An object of the subject matter of the present invention is to provide a method to identify  M. tuberculosis  using the restriction enzymes NruI and BamHI. 
         [0009]    Another object of the subject matter of the present invention is to provide a method to identify  M. tuberculosis  which is simple, cost effective, rapid, highly sensitive and specific. 
         [0010]    Another object of the subject matter of the present invention is to provide a method to differentiate between  M. tuberculosis  and Non Tuberculous mycobacteria. 
         [0011]    Another object of the subject matter of the present invention is to provide a kit to identify  M. tuberculosis.    
         [0012]    To achieve the above purposes, the present invention identifies  M. tuberculosis  in a subject by the following method:
       isolating chromosomal DNA from a biological sample   conducting a real time PCR to obtain an amplicon   digestion of said amplicon separately with restriction enzymes NruI and BamHI to obtain a digestion product   separating said digestion product into its constituents; and   analyzing the restriction enzyme digestion pattern       
 
         [0018]    The present invention also relates to a kit to identify  M. tuberculosis comprising  
       restriction enzymes NruI and BamHI; and   a manual for providing guidelines for identification of  M. tuberculosis          
 
         [0021]    The subject matter will now be described in detail with reference to accompanying examples. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0022]    The aforementioned aspects and other features of the present invention will be explained in the following description, taken in conjunction with the accompanying drawings, wherein: 
           [0023]      FIG. 1A  depicts the digestion of PCR amplicons of reference strains of  Mycobacterium  Spp. with NruI. Digestion of PCR amplicons of reference strains of  Mycobacterium  spp. with NruI. Lane 1, 50-bp DNA marker. Lanes 2 to 17, restriction digests of H37Rv,  M. bovis, M. bovis  BCG,  M. microti, M. smegmatis, M. simiae, M. xenopi, M. vaccae, M. malmoense, M. kansasii, M. fortuitum, M. avium, M. intracellulare, M. gordonae, M. phlei , and  M. terrae , respectively. Lane 18, 50-bp marker. 
           [0024]      FIG. 1B  depicts the digestion of PCR amplicons of reference strains of  Mycobacterium  Spp. with BamHI. Digestion of PCR amplicons of reference strains of  Mycobacterium  spp. with BamHI. Lane 1, 50-bp DNA marker. Lanes 2 to 17, restriction digests of H37Rv,  M. bovis, M. bovis  BCG , M. microti, M. smegmatis, M. simiae, M. xenopi, M. vaccae, M. malmoense, M. kansasii, M. fortuitum, M. avium, M. intracellulare, M. gordonae, M. phlei , and  M. terrae , respectively. Lane 18, 50-bp marker. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0025]    The present invention is explained in detail below. 
         [0026]    The inventors have developed a novel PCR Restriction Analysis (PRA) of the hsp65 gene for rapid screening of samples for  M. tuberculosis  complex (MTBC). Restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to make differentiation between MTBC and Non Tuberculous Mycobacteria (NTM) simple. The NruI/BamHI PRA is simple as it does not require any elaborate analyses. It is cost effective, rapid, highly sensitive and specific and did not require technical expertise. The assay could, therefore, be used as a simple screening test to differentiate MTBC from NTM in peripheral laboratories. 
         [0027]    PRA techniques have been developed for several mycobacterial genes, such as hsp65, the 16S-23S rDNA spacer and rpoB. However, most of these techniques require the use of an algorithm to identify the  Mycobacterium  species. Also, the formation of short restricts or small differences between bands needs the use of Nusieve or Metaphor agarose which are expensive ; or polyacrylamide gel, which is difficult to handle, thus making the assay technically demanding. The inventors of the present invention have developed a simple PRA technique to differentiate between MTBC and NTM, which does not require technical expertise and could be used as a screening assay in peripheral laboratories in tuberculosis endemic areas. It was cost effective, rapid, highly sensitive and specific. The assay could, therefore, be used as a simple screening test to differentiate MTBC from NTM in peripheral laboratories. 
         [0028]    The method is used even in small laboratories to identify  M. tuberculosis , and also to differentiate it from Non tuberculous mycobacteria. Non tuberculous mycobacteria (NTM) are ubiquitous and can be present as contaminants. They can also cause disease, especially in immuno-compromised patients. Routine smear examination cannot differentiate between MTBC and NTM. The present PRA method was evolved to make differentiation between MTBC and NTM easy not only in cultures, but also in clinical samples like sputum. 
         [0029]    In order that this invention may be better understood the following examples are described for illustrative purposes only. The examples illustrate the present invention and are not intended to limit it in spirit or scope. 
       EXAMPLES 
     Materials and Methods  
       [0030]    Clinical Isolates: A total of 310 clinical isolates were obtained from clinically suspected patients of pulmonary tuberculosis. The patients were adults ≧18 years old and were not co-infected with HIV. The clinical isolates were subjected to biochemical identification using niacin, nitrate reduction and semi-quantitative catalase tests by the standard procedure. 
         [0031]    Reference Strains: In addition to the clinical isolates of  Mycobacterium  sp., 24 reference i.e. strains  Mycobacterium tuberculosis  (H37Rv),  Mycobacterium Bovis  (ATCC 19210T),  Mycobacterium microti  (ATCC 25584),  Mycobacterium avium  (MTCC, IMTECH, Chandigarh, India),  Mycobacterium intracellulare  (ATCC 13950),  Mycobacterium gordonae  (ATCC 14470),  Mycobacterium fortuitum  (ATCC 6841),  Mycobacterium kansasii  (ATCC 21982),  Mycobacterium phlei  (ATCC 11758),  Mycobacterium smegmatis  (ATCC 19420),  Mycobacterium terrae  (ATCC 15755),  Mycobacterium vaccae  (ATCC 15483),  Mycobacterium malmoense  (ATCC 29571),  Mycobacterium xenopi  (ATCC 19250),  Mycobacterium simiae  (ATCC 25275T),  Nocardia brasiliensis  (ATCC 19296),  Nocardia asteroides  (ATCC 19247),  Escherichia coli  (ATCC 35218),  Klebsiella pneumoniae  (ATCC 13883),  Pseudomonas aeruginosa  (ATCC 33348),  Enterococcus faecalis  (ATCC 19433),  Staphylococcus aureus  (ATCC 25923),  Haemophilus influenzae  (ATCC 35891) and  Streptococcus pneumoniae  (ATCCBAA-255) were also included in the study. 
         [0032]    DNA Extraction from cultures and spiked sputum sample: Chromosomal DNA was extracted from the clinical mycobacterial isolates, the reference strain H37Rv and the reference non-tuberculous mycobacterial strains by the CTAB method as described previously. DNA was extracted from cultures of bacteria other than  Mycobacterium  sp. by boiling. Briefly, a loopful of mycobacterial growth was transferred to a micro-centrifuge tube containing 100 μl of 1% TritonX-100 and 50 μl of sterile double distilled water. The suspension was vortexed and boiled at 100° C. for 30 min. The suspension was centrifuged at 8000 rpm for 10 min and the clear supernatant containing mycobacterial DNA was taken for PCR. 
         [0033]    A smear negative sputum sample was spiked with serial dilutions of H37Rv to test the lower limit of detection with the assay. DNA was extracted from the spiked sputum samples as described earlier. Briefly, equal amounts of sputum sample and 4% NaOH were mixed and incubated for 10 min at 37° C. The mixture was centrifuged at 14000 rpm for 5 min and the pellet thus obtained washed three times with PBS (pH-7.4). The pellet was finally suspended in 1% Triton X-100 and the suspension boiled at 100° C. for 30 min. The lysate was centrifuged at 8000 rpm for 10 min and the supernatant collected for PCR. 
       Development of the Assay 
       [0034]    Sequences of thirty four mycobacterial species were obtained from GenBank; and target (Sequence ID No. : 1) HSP N3: 5′-AAGAAGTGGGGTGCCCCC-3′ and (Sequence ID No. 2) antisense HSP N4: 5′-CTTGGTCTCGACCTCCTTG-3′ primers specific for hsp65 of the mycobacterial species were designed. Restriction enzymes compatible with the 300 by region of hsp65 gene of MTBC were screened by the BioEdit software so that the restricted bands were large enough to be visualized on agarose gel. Search for restriction enzymes was made so as to obtain only two bands on digestion with the enzymes to make differentiation of MTBC with NTM simple. Restriction enzymes NruI and BamHI were selected for further experiments since both the enzymes restricted the 300 by region of hsp65 gene of MTBC, whereas the NTM were restricted by either NruI or BamHI but not both. 
         [0035]    PCR amplification 
         [0036]    Amplification of 310 cultures of  Mycobacterium  sp., 24 reference strains and sputum sample spiked with varying concentrations of H37Rv was performed with the primer set HSP N3 and HSP N4, amplifying a 300 by region of the hsp65 gene. 5 μl of the extracted DNA or 7 μl of the supernatant collected from processing the spiked sputum sample were used as template for PCR. The 30 μl PCR reaction mix consisted of 10 pM of each primer, 200 μM of each deoxynucleoside triphosphate, 1.5 mM MgCl2 and 0.8 U Taq polymerase (Biotools, B&amp;M Labs, Madrid, Spain). The thermal profile consisted of an initial denaturation for 10 min at 94° C., followed by 40 cycles of denaturation at 94° C. for 30s, annealing at 60° C. for 30s and primer extension at 72° C. for 1 min. Final extension was performed at 72° C. for 10 min. The PCR product was analyzed by electrophoresis on a 1.5% agarose gel along with a 50 by DNA ladder used as a marker. 
         [0037]    Positive and Negative Controls Used in the PCR Assay 
         [0038]    Each PCR run contained a positive control (H37Rv DNA) and a negative control (double distilled water). For PCR directly from spiked sputum samples, the samples were also tested for the presence of the human β-actin gene as described earlier (6). This served as an internal control in each PCR reaction. 
         [0039]    Restriction Analysis with NruI and BamHI 
         [0040]    Restriction digestion of the 300 by product was carried out with 1 U each of NruI and BamHI in separate tubes. Restriction digestion was carried out with 10 μl of the amplicon at 37° C. for 2 hours. The products were electrophoresed on 2.5% agarose gel with a 50 by DNA marker. 
         [0041]    PRA using Sau96I and CfoI 
         [0042]    To test the efficacy of the assay and confirm the test, 150 clinical mycobacterial isolates were also tested by PRA using the enzymes Sau96I and CfoI as previously described. 
         [0043]    Sequencing 
         [0044]    PCR products obtained by amplification of the hsp65 gene with primers HSP N3 and HSP N4 of a subset of clinical isolates (n=53) were subjected to sequencing by an ABI Automated Sequencer (Ocimum Biosolutions, Bangalore, India). Sequences were identified by similarity using Blastn available at NCBI. Species identification was confirmed if 97% match was achieved with any sequence deposited in the database, according to the criteria proposed by McNabb et al. 
         [0045]    Sensitivity of the Assay when Used Directly on Clinical Samples 
         [0046]    With the intent to identify  M. tuberculosis  directly in clinical specimens, the lower limit of detection of the assay using primers HSP N3 and HSP N4 was estimated. The sensitivity of the assay was tested on a smear negative sputum sample obtained from a patient not suffering from tuberculosis. The sample was spiked with serial dilutions of H37Rv (21) to obtain a final concentration of 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5  and 10 6  organisms/μl. DNA was extracted from the spiked sputum samples as described above. The suspensions were subjected to PCR with primer sets HSP N3 and HSP N4. PRA was performed on the amplicons obtained with restriction enzymes NruI and BamHI as described previously. 
         [0047]    Statistical Analysis 
         [0048]    Kappa value was determined to find out the agreement between NruI/BamHI and Sau96I/CfoI assays by using GraphPad software (GraphPad, La Jolla, Calif.). 
       Results 
     Identification of Mycobacteria by Biochemical Reactions. 
       [0049]    All the clinical isolates were subjected to biochemical identification by Niacin, Nitrate reduction and semi-quantitative catalase tests. Of the 310 isolates, 236 were identified as  M. tuberculosis , whereas, 8 isolates were identified as NTM. However, 66 isolates could not be identified definitively on the basis of biochemical reactions. 
         [0000]    Identification of Mycobacteria by hsp65 PRA Using the Restriction Enzymes NruI and BamHI. 
         [0050]    The DNA obtained from 310 cultures of  Mycobacterium  sp. and 15 mycobacterial reference strains were used to amplify a 300 by region of the hsp65 gene using the primers HSP N3 and HSP N4. All mycobacterial isolates were amplified by the primers. PCR with primers HSP N3 and HSP N4 was also carried out with all the non-mycobacterial reference strains (viz.  Nocardia brasiliensis, Nocardia asteroides, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Haemophilus influenzae  and  Streptococcus pneumoniae ) obtained from ATCC and MTCC (IMTECH, Chandigarh). However, none of the non-mycobacterial species were amplified. 
         [0051]    Restriction enzymes compatible with the 300 by region of hsp65 were screened. The enzyme NruI was found to restrict the amplified sequences of  M. tuberculosis  complex at TCG/CGA giving two bands (121 by and 179 bp) ( FIG. 1A ), while BamHI restricted the same sequences at G/GATCC giving two bands (66 by and 234 bp) ( FIG. 1B ). 
         [0052]    We labeled the restricted pattern with Nrul as “A” and the unrestricted pattern as “a”. The restricted pattern of BamHI was labeled as “B” and the unrestricted pattern was labeled as “b” (Table 1).  M. tuberculosis, M. Bovis  and  M. microti  were the only mycobacterial species amongst all the species tested which were restricted by both NruI and BamHI (restriction pattern “AB”) (Table 2). Of all the culture isolates tested, 295 were identified as  M. tuberculosis  complex by NruI/BamHI PRA, including 59 of the 66 isolates that had not been identified definitively on the basis of biochemical reactions. The remaining 7/66 isolates that could not be identified definitively by conventional methods were determined to be NTM by NruI/BamHI PRA. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 Restriction pattern of 
                   
               
               
                   
                 PCR amplicon of hsp65 gene 
               
             
          
           
               
                 Restriction Enzyme 
                 Restricted 
                 Not restricted 
               
               
                   
               
               
                 NruI 
                 A 
                 B 
               
               
                 BamHI 
                 a 
                 b 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 RESTRICTION PATTERN OF  MYCOBACTERIUM   
               
               
                 SP. USING NRUI AND BAMHI. 
               
             
          
           
               
                   
                 Restriction pattern 
                   
               
             
          
           
               
                   
                   Mycobacterium  sp. 
                 NruI 
                 BamHI 
               
               
                   
                   
               
               
                   
                 H37Rv 
                 A 
                 a 
               
               
                   
                 
                   M. bovis 
                 
                 A 
                 a 
               
               
                   
                 
                   M. microti 
                 
                 A 
                 a 
               
               
                   
                 
                   M. smegmatis 
                 
                 A 
                 b 
               
               
                   
                 
                   M. simiae 
                 
                 A 
                 b 
               
               
                   
                 
                   M. xenopi 
                 
                 B 
                 a 
               
               
                   
                 
                   M. vaccae 
                 
                 A 
                 b 
               
               
                   
                 
                   M. melomoense 
                 
                 A 
                 b 
               
               
                   
                 
                   M. kansasii 
                 
                 B 
                 b 
               
               
                   
                 
                   M. fortuitum 
                 
                 B 
                 b 
               
               
                   
                 
                   M. avium 
                 
                 B 
                 b 
               
               
                   
                 
                   M. intracellularae 
                 
                 B 
                 b 
               
               
                   
                 
                   M. gordonae 
                 
                 B 
                 b 
               
               
                   
                 
                   M. phlei 
                 
                 B 
                 a 
               
               
                   
                 
                   M. terrae 
                 
                 B 
                 b 
               
               
                   
                   
               
             
          
         
       
     
       Testing the Efficacy of NruI/BamHI PRA 
       [0053]    To test the ability of the PRA using restriction enzymes NruI and BamHI in correctly differentiating between MTBC and NTM, 150 clinical isolates were subjected to PRA using the enzymes Sau96I and CfoI. These included all the isolates identified to be NTM. Of the 150 mycobacterial isolates, 135 were identified as MTBC by both the methods. Of the 15 isolates identified as NTM by using the enzymes NruI and BamHI, all were confirmed to be NTM by using Sau96I and CfoI also. 
       Sequencing 
       [0054]    Of the 53 isolates sequenced, thirty eight had been restricted by both NruI and BamHI and were identified as  M. tuberculosis  on sequencing, thus confirming our results. Of the remaining 15 clinical isolates sequenced, all had been determined to be NTM by PRA using NruI/BamHI as well as Sau96I/CfoI. The isolates were identified to be  M. avium -intracellulare (n=9),  M. fortuitum  (n=3) or  M. abscessus  (n=3) by sequencing. The NruI/BamHI assay was thus, 100% concordant with sequencing (Kappa value 1). 
       Sensitivity of the Assay 
       [0055]    To detect the sensitivity of PRA using primers HSP N3 and HSP N4 and enzymes NruI and BamHI directly on sputum samples, DNA was extracted from serial dilutions of H37Rv used to spike a smear negative sputum sample to obtain a final concentration of 10°, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , and 10 6  organisms/μl and used as template for hsp65 PCR. PRA was performed, on the amplicons obtained, with restriction enzymes NruI and BamHI. The lower limit of identification with BamHI was 10 organisms/μl, however, a reliable PCR restriction analysis with both NruI and BamHI was only possible down to a concentration of 10 2  organisms/μl. 
         [0056]    The various embodiments described above can be combined to provide further embodiments. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. 
         [0057]    Although the disclosure of system and method has been described in connection with the embodiment of the present disclosure illustrated in the accompanying drawings and examples, it is not limited thereto. It will be apparent to those skilled in the art that various substitutions, modifications and changes may be made thereto without departing from the scope and spirit of the disclosure.