Claims:1. A multiplex PCR assay for detecting a status of infection of Mycobacterium Tuberculosis Complex (MTBC) or Mycobacterium Other Than Tuberculosis (MOTT) in a sample, comprising isolating DNA and mRNA OR nucleic acids from the sample ; optionally performing reverse transcription on isolated mRNA to obtain cDNA, pooling the isolated DNA and the cDNA together and amplifying the pooled DNA simultaneously for detection of IS6110 through a first primer set, detection of 85B cDNA through a second primer set, detection of 16S r RNA through a third primer set , and determining the state of the infection of MTBC/MOTT in accordance with following:
IS6110 DNA 85B cDNA 16S r RNA Conclusion
Positive Positive Positive Viable MTBC detected
Positive Negative Positive Dormant MTBC detected
Positive Negative Negative Dead MTBC detected
Negative Negative Positive MOTT detected
Negative Negative Negative MTBC/MOTT not detected
(Complete recovery)
Negative Negative Negative No Amplification

2. The method of claim1, wherein, the said Mycobacterium Tuberculosis Complex (MTBC) comprises Mycobacterium tuberculosis Complex, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae and Mycobacterium mungi.
3. The method of claim1 wherein, the Mycobacterium other than tuberculosis (MOTT) comprises all mycobacteria other than MTBC.
4. The method of claim1, wherein, the sample comprises blood, mucus, pus, Bronchoalveolar lavage, tissue, cerebrospinal fluid and/or any biological fluid.
5. The method of claim1, wherein, the said status of infection is determined by state of bacilli as being viable or dormant or dead.
6. The method of claim1, for detection of Mycobacterium tuberculosis Complex (MTBC)or Mycobacterium Other Than Tuberculosis (MOTT),characterised in qualitative and quantitative measurement of mycobacteria in a sample further characterised in that the method is highly effective in qualitative and quantitative measurement of viable, dormant and dead mycobacterium.
7. The method of claim1, wherein, the said status of infection is active tuberculosis or latent tuberculosis or complete recovery.
8. The method of claim 1, wherein, melting curve analysis is used to distinguish detected bacilli as viable or dormant or dead.
, Description:DETAILED DESCRIPTION OF THE INVENTION

As required, detailed embodiment are disclosed herein; however, it is to be understood that the disclosed embodiment are merely exemplary, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the present embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment herein belong. The specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention.
The term “primer” used herein refers to short pre-existing polynucleotide chain to which new deoxy ribonucleotides can be added by DNA polymerase.
The term “ multiplex PCR “ used herein, refers to a Polymerase Chain Reaction in which more than one DNA sequence can be amplified simultaneously by using multiple sets of primers within a single PCR mix giving amplicons of varying sizes specific to those DNA sequences.
The term” MTBC” refers to Mycobacterium Tuberculosis Complex, which includes a genetically related group of Mycobacterium species that causes tuberculosis in humans. Alternately, “MOTT” refers to Mycobacterium Other Than Tuberculosis, which includes mycobacteria that do not cause tuberculosis, but may cause other pulmonary diseases in humans.
”mRNA” refers to messenger RNA, which conveys genetic information from DNA by translating into a protein. The presence of mRNA in a cell is indicative of active protein synthesis and hence growth & viability.
“16S r RNA” refers to 16 S ribosomal RNA, which participates in translation process.The term “viable” used herein, refers to actively dividing mycobacterium bacilli. The term “dormant” used herein refers to live but non-dividing mycobacterium bacilli. The term “dead” used herein refers to non-living mycobacterium bacilli. The term “active” used herein refers to infection caused by viable bacilli. The term “latent “used herein refers to infection caused by dormant bacilli that may become “active” anytime in future.

The embodiment herein provide a multiplex PCR assay for detecting a state of infection of Mycobacterium tuberculosis Complex (MTBC)or Mycobacterium Other Than Tuberculosis (MOTT) in a sample. The multiplex PCR assay includes isolating DNA and total RNA OR nucleic acids from the sample ; optionally performing reverse transcription on isolated mRNA to obtain cDNA, pooling the isolated DNA and the cDNA together and amplifying the pooled DNA simultaneously for detection of IS6110 through a first primer set, a for detection of 85B cDNA through a second primer set, detection of 16S r RNA through a third primer set , and determining the state of the infection of MTBC/MOTT in accordance with following table. :

IS6110 DNA 85B mRNA 16S r RNA Conclusion
Positive Positive Positive Viable MTBC detected
Positive Negative Positive Dormant MTBC detected
Positive Negative Negative Dead MTBC detected
Negative Negative Positive MOTT detected
Negative Negative Negative MTBC/MOTT not detected
(Complete recovery)
Negative Negative Negative No Amplification

The detection of IS6110, 85B antigen and 16S r RNA indicates presence of viable MTBC bacilli; detection of IS6110 and 16S r RNA indicates presence of dormant MTBC bacilli; detection of IS6110 only indicates presence of dead MTBC bacilli; detection of 16SrRNA only indicates presence of MOTT; and detection of viable MTBC bacilli is termed as active status of infection; detection of dormant MTBC bacilli is termed as Latent status of infection; and detection of dead MTBC bacilli is termed as complete recovery status .

The embodiment herein provides a method of detection of mycobacteria in a biological sample, for the diagnosis of TB and to assess the status of MTB infection, if any, in an individual, so as to have correct prognosis that enables effective treatment regime. The present methods/assays/kits may be employed to ascertain whether an individual is suffering from active TB and needs treatment or has latent TB and may acquire active TB in future or has been cured completely following a treatment. Alternately, the present methods/assays/kits may help clinicians monitor their patients accurately during the long treatment period and evaluate success or failure of the treatment intermittently and towards the end with utmost certainty.

The methods, compositions, and kits provided herein provide diagnostically relevant information as well as a basis for treatment of patients who exhibit pulmonary/ extra pulmonary infections that may be due to mycobacterial infections. In an embodiment, the assays provided herein, determine whether a sample contains mycobacterium. In another embodiment, the assays provided herein, determine whether a sample contains mycobacterium other than tuberculosis. In yet another embodiment, the assays provided herein determine whether a pulmonary/ extra pulmonary infection contains mycobacterium.
The assays provided herein may determine whether a mycobacterium in a sample or infection is a member of the Mycobacterium Tuberculosis Complex (MTBC) or Mycobacterium Other Than Tuberculosis (MOTT). In a preferred embodiment, the assays provided herein determine if a sample contains, or an infection is due to, viable bacteria belonging to MTBC or MOTT group. In another preferred embodiment, the assays provided herein determine if a sample contains viable or dormant or dead bacilli. In yet another preferred embodiment, the assays provided herein determine if mycobacterium infection is active or latent. In yet another preferred embodiment, the assays provided herein determine if a patient undergoing anti-TB treatment is free of active TB bacilli or not. In yet another embodiment, the assays or methods provided herein determine the success of anti-TB treatment by detecting viable or dead bacilli.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages may mean ±1%. In this application, the use of “or” implies “and/or” unless stated otherwise. Furthermore, the use of the term “including,” as well as other forms, such as “includes” and “included”, is not limited. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

The present method includes a multiplex PCR assay to detect IS6110, 16S r RNA and 85B cDNA simultaneously from a DNA pool isolated from a patient sample. The patient sample includes any biological fluid or specimen such as blood mucus, pus, tissue, BAL (Broncho alveolar lavage), cerebrospinal fluid, urine or any other biological sample. IS6110 is an insertion element that is found exclusively within the members of Mycobacterium Tuberculosis Complex (MTBC) making it an important diagnostic tool in differentiation of MTBC species from other Mycobacteria. Additionally, this element is present in multiple copies at different locations within Mycobacterium genome, thereby enhancing detection signal.16S r RNA is found to be conserved and highly specific to all Mycobacteria including MTBC and MOTT. The present detection or diagnosis method utilizes these two genes towards identification of mycobacteria as well as for differentiation between MTBC and MOTT by means of a multiplex PCR that detects and amplifies IS6110 and 16 S rRNA genes within the DNA isolated from the biological sample of the patient. 85B antigen is one of the most abundantly expressed proteins in broth cultures and human mononuclear phagocytes. Bacterial mRNA being highly short lived with an approximate half- life of five minutes, detection of 85B mRNA by means of quantitative RT-PCR serves as reliable marker for mycobacterial viability.

As illustrated in Figure 1, DNA and total RNA are extracted from patient sample by standard protocols. mRNA is isolated from total RNA and reverse transcribed to obtain cDNA. The isolated DNA and cDNA are put together as a single pool, which is further subject to Multiplex PCR amplification. Primers for amplification of IS6110 gene, 16S r RNA gene and 85B mRNA are developed using methods known in the art. Actin gene amplification is carried alongside as a positive control in the reaction. Multiplex PCR amplification is performed at standardized PCR conditions following which amplified PCR products are detected by gel electrophoresis or hybridization or melting curve analysis or other methods well known in the art.

In an embodiment, melting curve analysis is performed by heating the PCR reaction mixture slowly up to 97°C, which causes melting of double-stranded DNA and a corresponding decrease of SYBR Green I fluorescence. The instrument continuously monitors this fluorescence decrease and displays it as melting peaks. Each melting peak represents the characteristic melting temperature (Tm) of a particular DNA product thereafter to prove that only desired PCR products have been amplified. External positive controls (MTC/MOTT) and negative controls are run along with each run for confirmation of the quality of assay performance. Housekeeping gene Actin serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition.

Fluorescence peaks obtained in channels are compared to expected melting temperatures (Tm) of desired amplicons i.e. Actin at Tm 85± 0.1 (positive control), IS6110 at Tm ±0.1, 16S r RNA at Tm 85.2±0.1 and 85B at Tm 88.2±0.2 respectively. Positive signal for actin only indicates absence of mycobacterium (MTBC and MOTT both), whereas, positive signal for actin and 16 S r RNA only, indicate presence of MOTT. Positive signal in all channels is indicative of viable MTBC, whereas absence of 85B peak is indicative of dormant MTBC bacilli. Absence of positive signal in 85 B and 16 S r RNA and positive signal in IS6110 channel indicates presence of MTBC DNA belonging to dead bacilli.

The embodiment herein include a kit for qualitative and quantitative measurement of mycobacteria in a sample further characterised in that the kit is highly effective in qualitative and quantitative measurement of viable, dormant and dead mycobacterium. In one of the embodiments, the method of measurement comprises extraction of RNA and/or DNA from sample selected from but not limited to sputum, Broncho alveolar lavage (BAL), CSF, body fluids (pleural, pericardial, gastric, ascitic, seminal), blood, tissue, pus, urine, paraffin block etc. In one of the embodiment, biomarkers used for detection of mycobacteria includes but not limited to at least one conservative region of r RNA, mRNA, cDNA, 85B antigen, insertion element IS6110, 16S rRNA and like. In another embodiment, biomarkers are used alone or in combinations in same reaction kit. In yet another embodiment, real time detection technology is used. In further embodiment one set of experiment involves optional use of positive controls for example but not limited to MTBC/MOTT.
In an embodiment, the assays/ kits provided herein, detect MTBC/MOTT in specimen based on detection of specific nucleic acid sequences. In another embodiment, the assay /kit provided herein, detects 85 B mRNA, presence of which confirms viable MTBC. In a further embodiment, the assay /kit provided herein, detects IS6110 DNA, presence of which confirms MTB complex. In yet another embodiment, the assay /kit provided herein, detects 16rRNA, presence of which, confirms MTB complex which could be viable or dormant or dead.
It is to be understood that while the presented subject matter has been described in conjunction with the embodiments described herein, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions of the invention. The examples are intended as non-limiting examples of the invention. While efforts have been made to ensure accuracy with respect to variables such as amounts, temperature, etc., experimental error and deviations should be taken into account. Unless indicated otherwise, parts are parts by weight, temperature is degrees centigrade, and pressure is at or near atmospheric. All components were obtained commercially unless otherwise indicated.
Example 1

Pre-Treatment of clinical sample before DNA/RNA extraction
1.1 Mucoid specimen - (sputum, viscous body fluid, BAL, pus, etc.)
The sample was transferred to 15ml falcon tube (not exceeding 1/5th of the volume of the tube) and equal volume of freshly prepared NALC-NaOH digestant was added and mixed by gently vortexing until liquefied. Samples were then left at room temperature for 15 minutes, following which volume was adjusted to 15 ml with sterile water. Thereafter, the contents of the tube were mixed well and centrifuged at 3000 rpm for 15 minutes. Further, the supernatant was discarded carefully and the pellet was re-suspended in 200 µl sterile Phosphate buffer saline. This was then used for DNA/RNA extraction.
1.2 Tissue specimen
Solid tissue was minced in few drops of 0.1M sterile phosphate buffer saline (PBS) using a surgical blade followed by centrifugation at 2,500 rpm for 10 minutes. The supernatant was discarded and the pellet was re-suspended in 1 ml PBS. This was then used for DNA/RNA extraction.

1.3 Non-mucoid specimen (Body fluids, CSF, etc.)
2-3 ml of specimen was transferred to a 50-ml plastic centrifuge tube and an equal volume of SDS-NaOH digestant solution was added to it. The mixture was vortexed gently and incubated at room temperature for 15 minutes. Thereafter, the contents of the tube were mixed well and centrifuged at 3000 rpm for 15 minutes. Further, the supernatant was discarded carefully and the pellet was re-suspended in 200 µl sterile Phosphate buffer saline. This was then used for DNA/RNA extraction.

1.4 Urine specimen
The pH of urine was checked and neutralized with 1N NaOH/1N HCl, if required. The specimen was then centrifuged at 3,500 rpm for 10 minutes and the supernatant was discarded leaving about 1 ml sample. Further, the pellet was re-suspended by adding an equal volume of SDS-NaOH digestant solution and gently vortexed and incubated at room temperature for 15 minutes. Further, 15 ml of sterile distilled water was added and centrifuged at 4,000 rpm for 15 minutes. The supernatant was discarded carefully; ensuring that the pellet was not disturbed and washing was repeated 3 times. After the final wash, the pellet was re-suspended in 1 ml PBS. 450 µl of this specimen was used for DNA/RNA extraction.
Example 2
Isolation of Genomic DNA & Total RNA

Genomic DNA & Total RNA were isolated according to standard protocols. mRNA was further isolated and reverse transcribed to obtain cDNA. Genomic DNA and cDNA were pooled together into a single pool for next step.
Example 3
Multiplex PCR amplification of target genes
Multiplex PCR was performed using primers specific for the IS6110, 85B cDNA and 16 S r RNA. Primers for actin gene were taken alongside as a positive control.
Example 4
PCR product analysis by Melting Curve analysis

Amplification products were detected by melting curve analysis for which the reaction mixture was slowly heated to 97°C.Fluorescence peaks were obtained based on the melting temperatures of the amplicons formed. Table 1 below shows the melting temperatures of the desired products and Table 2 shows the basis of interpretation of the results obtained.
Table1.
Gene Melting Temperature
Actin 85± 0.1
85 B antigen 88.2±0.2
IS6110 ±0.1
16S r RNA 85.2±0.1
Table2.
Actin (positive control) IS6110 DNA 85B mRNA 16S r RNA Conclusion
Positive Positive Positive Positive Viable MTBC detected
Positive Positive Negative Positive Dormant MTBC detected
Positive Positive Negative Negative Dead MTBC detected
Positive Negative Negative Positive MOTT detected
Positive Negative Negative Negative MTBC/MOTT not detected(complete recovery)
Negative Negative Negative Negative No Amplification