Patent Grant
10597734
This application claims priority to International Patent Application No. PCT/IB2014/066469, filed Dec. 1, 2014, which claims priority to Indian Patent Application No. 5572/CHE/2013, filed Dec. 3, 2013, both of which are hereby incorporated herein by reference in their entireties.
The application is related to novel signature sequences for diagnosis of Mycobacterium tuberculosis in clinical samples. These signature sequences have the ability to differentiate Mycobacterium tuberculosis DNA from other mycobacterial species by PCR with 100% specificity and very high sensitivity.
Tuberculosis (TB) is a major global health problem with an alarming rate of mortality associated with it. It is one of the leading infectious diseases caused by bacteria taking one human life every 15-20 seconds globally. Estimates of 2011 reveal that there are almost 9 million new cases and 1.4 million TB deaths (Global Tuberculosis Report 2012, WHO 2013). The disease is caused by Mycobacterium tuberculosis, a member of the genus Mycobacterium, while in a few cases Mycobacterium bovis has been reported to be the causal organism. More than 100 mycobacterium species are known and among them only a few are pathogenic for humans.
Conventional diagnostic methods include examination of sputum smear under a microscope for acid-fast mycobacteria and radiological reading of lungs. However, in most cases the sputum smear examination turns out to be negative for the bacteria due to early stages of infection and lung changes are not readily visible on an x-rays until several months into the infection.
Diagnosis of mycobacteria-related disease poses difficulties for several reasons which have been recognized by researchers and clinicians over the years. Firstly, these bacteria are in small numbers and are already at a contagious stage when they become detectable by conventional methods. Pulmonary disease caused by different mycobacteria are not readily detectable clinically or radiologically. Detection of organism in culture achieves 100% specificity but the growth of mycobacteria in culture takes about 3-6 weeks (Bates et al, Am. Respir. 134, 415-417, 1986) thus making the process time-consuming. In addition, repeated cultures may be required to ensure success.
Several molecular tests for tuberculosis have been developed in the past. These include the Gen-probe ‘Amplified mycobacterium direct test’ by Abbe et al (J. Clin. Mincrobiol. 31, 3270, 1993), ligase chain reaction (LCR) (J. Clin. Microbiol. 35, 2424, 1997), PCR based Roche Amplicor TB test (J. Clin. Microbiol. 33, 1832, 1995) and IS6110 based detection (J. Clin. Microbiol. 28, 2668, 1990).
U.S. Pat. No. 5,168,039 discloses the IS6110 based detection of M. tuberculosis wherein a repetitive DNA segment specific for members of M. tuberculosis complex is used for the diagnosis. While IS6110 based detection system has been shown to have high level of specificity, there are also reports on false positive detections of 3 to 20% making it unreliable (J. Clin. Microbiol. 32, 277, 1994). In addition, lack of IS6110 sequence in some M. tuberculosis strains may also limit the use of the same for diagnostic tests routinely (Tuber. Lung Dis. 76, 550, 1995). U.S. Pat. No. 7,638,309 provides detection of mycobacteria in clinical specimens in the intergenic region between methyl mycotic acid synthase genes mmaA1 and mmaA2 and the flanking region in mmaA1 and mmaA2 genes.
Thus, it appears that there is a paucity of simple, rapid and reliable tests that can specifically detect M. tuberculosis during early stages of the disease. The present invention has identified ‘signature sequences’ that can differentiate M. tuberculosis from a are number of other mycobacterial DNA. These ‘signature sequences’ are used in detection of early disease in clinical samples of patients.
The invention provides novel signature sequences for diagnosis of Mycobacterium species (sps) in clinical samples with 100% specificity and a very high degree of sensitivity.
In one aspect, the invention provides a nucleotide sequence capable of selectively detecting pathogenic Mycobacterium sps using oligonucleotide primers corresponding to the signature sequence selected from SEQ ID NO: 1, 2, 3 or 4.
In another aspect the invention provides a method of detecting pathogenic mycobacterium sps in a clinical sample, said method comprising the steps of:
a. removal of contaminants from the clinical sample by conventional methods;
b. extraction of genomic DNA from the contaminant-free clinical sample;
c. designing a set of specific oligonucleotide primers capable of specifically detecting SEQ ID NO: 1, 2, 3 or 4 for use in RT-PCR;
d. analyzing PCR product by electrophoresis or specific probe nucleotide sequence complementary to SEQ ID NO: 1, 2, 3 or 4.
The set of oligonucleotide primers of the invention are selected from
(v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv) or
(vi) a set of oligonucleotide primers comprising of sequence containing any 10 consecutive bases from one of the sequences selected from SEQ ID NO: 1, 2, 3 or 4.
In yet another aspect, the invention provides a kit for the detection of pathogenic mycobacterium sps in clinical samples, said kit comprising set of oligonucleotide primers selected from
(v) a set of oligonucleotide primers complementary to (i), (ii), (iii) or (iv) or
(vi) a set of oligonucleotide primers comprising of sequence containing any 10 consecutive bases from one of the sequences selected from SEQ ID NO: 1, 2, 3 or 4.
Furthermore, the invention provides a method of detecting pathogenic mycobacterium sps in a clinical sample wherein the sample is isolated from individuals vaccinated against tuberculosis.
It also provides a method of detecting pathogenic mycobacterium sps in a clinical sample wherein the sample is isolated from individuals treated against tuberculosis.
The present invention relates to detection of pathogenic Mycobacterium species using signature sequences SEQ ID NO: 1, 2, 3 or 4 with a high degree of sensitivity and 100% specificity.
In one embodiment, the invention provides novel DNA diagnostic markers for specific detection of Mycobacterium tuberculosis which causes tuberculosis.
A three-pronged approach was carried out to identify novel DNA diagnostic marker for detection of pathogenic mycobacterium sps, especially, Mycobacterium tuberculosis. First step provides an in-silico approach to identify and shortlist potential sequences of Mycobacterium, unique and exclusive to pathogenic mycobacterium sps, especially Mycobacterium tuberculosis. The criteria used for selection of the potential sequences are presented below which involves comparative proteomic analysis of 13 mycobacterium species:
The following bioinformatics process flow resulted in the identification of potential gene markers of the invention.
The above process flow resulted in the classification of the potential hits into class 1 and class 2. As per the present invention, class 1 are genes unique to the organism of interest based on the fact that they do not share protein domain and protein sequence identity of more than 20% and nucleotide sequence identity of more than 35% with any other organism in the selected organism list. In another embodiment, class 2 genes are those that do not share protein domain and protein sequence identity of more than 20% and nucleotide sequence identity of more than 35% with any other organism in the selected organism list.
Table 1 provides potential candidate genes carrying ‘signature sequences’ based on the bioinformatics process flow.
In one aspect, the invention functionally characterizes the potential ‘signature sequences (SS)’-carrying candidate genes based on functional information retrieved from Tuberculist (Tuberculosis (Edinb) 91, 7, 2011) and TB database (Nucleic Acids Research, 37, D499, 2009). Accordingly, the potential signature sequences can be functionally characterized into the following groups:
Based on the in-silico analysis, two Class 1 genes (Rv1507A and Rv1509) and two Class 2 genes (Rv1954A and Rv2231A) with homologs in Mycobacterium bovis BCG were selected as potential candidates (Table 2).
Mycobacterium tuberculosis H37Rv
Mycobacterium tuberculosis H37Rv
In a most preferred aspect, the signature sequences were designed keeping in view the diagnostic tool of RT PCR. These were short sequences amenable for PCR amplification from selected genes. The specific signature sequences, SS1 (Rv1507A), SS2 (Rv1509), SS3 (RV1954A) and SS4 (Rv2231A) of the invention are provided below. Homology search using NCBI nucleotide BLAST against the genus Mycobacterium was conducted on these signature sequences to confirm their uniqueness.
Mycobacterium tuberculosis H37Rv complete genome
Mycobacterium tuberculosis H37Rv complete genome
Mycobacterium tuberculosis H37Rv complete genome
For the purposes of PCR validation, the signature sequences SS1, SS2, SS3 and SS4 were selected and oligonucleotide primers were designed to generate corresponding specific PCR amplification products. Table 3 provides the set of designed oligonucleotide primers.
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis
In a preferred embodiment, pathogenic mycobacterium sps can be detected with 100% specificity following PCR using DNA isolated from clinical samples from patients who presented with clinical symptoms of the disease. In another embodiment, pathogenic mycobacterium sps is also detected using the above method in clinical samples isolated from individuals vaccinated against tuberculosis. In yet another embodiment, pathogenic mycobacterium sps is also detected using the above method in clinical samples isolated from individuals treated against tuberculosis.
Pathogenic mycobacterium sps, as provided in the invention, includes Mycobacterium tuberculosis and Mycobacterium bovis. More specifically, pathogenic mycobacterium sps represents Mycobacterium tuberculosis, the TB causing bacterium.
Clinical samples, as meant here, includes specimens such as blood, sputum, cerebrospinal fluid, gastric lavage, tissue biopsies and the likes thereof. PCR product can be easily visualized by any conventional method that can be readily recognized by a person skilled in the art such as electrophoresis.
Following Examples serve as a tool to illustrate the invention. However, it should in no way be considered to be limiting the invention.
Determination of Specificity and Sensitivity of Signature Sequences
Genomic DNA for PCR Amplification
Genomic DNA of Mycobacterium tuberculosis and 13 other mycobacterial species were used for testing the specificity of signature sequences using PCR. These include, M. avium subspecies paratuberculosis, M. smegmatis (ATCC19420), M. vaccae, M. marinum (ATCC927), M. chelonae (ATCC14472), M. flavescens (ATCC14474), M. fortuitum (ATCC6481), M. kansasii (ATCC12478), M. bovis (ATCC27294), M. bovis (BCG), M. avium (ATCC25291), M. gastri, M. indicuspranii.
PCR Reaction
The PCR reaction mixture (50 μl) consisted of 10×taqPCR buffer, 0.5 mmolMgCl2, 0.4 mmol dNTP, 10 pmol forward and reverse primers respectively, 4% DMSO and 1 Utaq DNA polymerase. The reaction conditions were the following: 95° C. for 5 minutes, followed by 35 cycles of 95° C. for 30 seconds, annealing temperature 50° C. for 30 seconds, 72° C. for 1 minute and finally 72° C. for 10 minutes. All PCR products were electrophoresed on 2% agarose gel with ethidium bromide staining.
The “signature sequences” were tested for their ability to differentiate Mycobacterium tuberculosis DNA from a large number of other mycobacterial DNA in PCR using primers complementary to these “signature sequences” as shown in Table 3. For this purpose, chromosomal DNA extracted from 13 mycobacterium species including human genomic DNA were tested by mycobacteruim genus-specific primers of the ‘signature sequences’. SS1 and SS2 were negative for all 13 mycobacterium species tested whereas SS3 and SS4 show positive PCR results only when M. bovis BCG genomic DNA was used as template.
Table 4 summarizes the specificity data resulting from PCR using specific primers of signature sequences SS1, SS2, SS3 and SS4.
Mycobacterium tuberculosis and M. bovis BCG
M. tuberculosis
M. bovis BCG
M. avium
M. smegmatis
M. vaccae
M. avium
M. chelonae
M. flori
M. fortuitum
M. kansasi
M. bovis
M. marinum
M. gastri
M. leprae
Furthermore, sensitivity analysis revealed that the signature sequences were highly sensitive in being able to detect <1 ng (100 pg) DNA as shown in
Evaluation of Mycobacterium tuberculosis-Specific Primer Pair Using Clinical Samples
A) Amplification of Signature Sequences from Patient Sputum Samples
Sputum samples were processed by the Universal Sample Processing (USP) method for DNA extraction as described by Chakravorty et al (J Clin Microbiol 43, 4357, 2005). DNA was isolated from the USP sediments by boiling in the presence of five volumes of solution containing 10% Chelex-100 resin, 0.03% triton X-100, and 0.3% Tween 20. The isolated DNA was stored at 20° c. and used for PCR assay.
PCR reaction was carried out using specific primers as given in Table 3.
The results show amplification of signature sequences in patient sputum sample (
B) Amplification of Signature Sequences from Patient Blood Samples
DNA from blood samples of tuberculosis patients were isolated as per the protocol described in van Helden et al (Isolation of DNA from Mycobacterium tuberculosis, Paul D. van Helden, Thomas C. Victor, Robin M. Warren, and Eileen G. van Helden)
The results show amplification of SS1 and SS3 as seen in
| Number | Date | Country | Kind |
|---|---|---|---|
| 5572/CHE/2013 | Dec 2013 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2014/066469 | 12/1/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/083056 | 6/11/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6183957 | Cole | Feb 2001 | B1 |
| 6294328 | Fleischmann et al. | Sep 2001 | B1 |
| 20030135026 | Reed et al. | Jul 2003 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20160355872 A1 | Dec 2016 | US |
