WO2021195794A1 - Procédé et système de diagnostic moléculaire pour la détection de différentes souches de mycobacterium tuberculosis - Google Patents

Procédé et système de diagnostic moléculaire pour la détection de différentes souches de mycobacterium tuberculosis Download PDF

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WO2021195794A1
WO2021195794A1 PCT/CL2020/050034 CL2020050034W WO2021195794A1 WO 2021195794 A1 WO2021195794 A1 WO 2021195794A1 CL 2020050034 W CL2020050034 W CL 2020050034W WO 2021195794 A1 WO2021195794 A1 WO 2021195794A1
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seq
primers
samples
sample
gene
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PCT/CL2020/050034
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Vívian D’AFONSECA DA SILVA FERREIRA
Ariel Domingo ARENCIBIA RODRÍGUEZ
María De Los Ángeles RECABAL SALAZAR
Elizabeth Catalina VALDÉS MUÑOZ
Dafne Alejandra REYES FARÍAS
Francisca Beatriz VALENZUELA SALGADO
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Universidad Católica Del Maule
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Priority to PCT/CL2020/050034 priority Critical patent/WO2021195794A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the present invention is framed in the area of clinical diagnosis and provides an in vitro diagnostic method for the detection of different strains of Mycobacterium tuberculosis in samples obtained from human tissues by means of PCR using a set of specific primers or primers characterized by be a sensitive and specific molecular diagnostic test.
  • a system and / or diagnostic kit related to said method is provided that uses the specific designed primers.
  • Tuberculosis is an infectious disease caused by strains of Mycobacterium tuberculosis and other species of the genus Mycobacterium. This disease is in sixth place among the 10 leading causes of death in the world.
  • vaccines, diagnostic tests and treatments for tuberculosis are not limiting the advance of this disease in various countries of the world, including Chile.
  • Factors that can increase the rate of tuberculosis infection in a population include: increase in HIV infections, increase in the number of homeless people (poverty and malnutrition), and drug-resistant strains of tuberculosis.
  • tuberculosis According to the World Health Organization (WHO), in 2016, 10.4 million people contracted tuberculosis and 1.7 million died (including 0.4 million people with HIV). More than 95% of tuberculosis deaths occur in low- and middle-income countries. Therefore, tuberculosis continues to be a public health problem worldwide.
  • WHO World Health Organization
  • End tuberculosis In WHO there is a program under development called “End tuberculosis” which seeks to control and eradicate the global epidemic by the year 2035, where countries affected by the presence of Mycobacterium tuberculosis must strengthen their national programs to combat this disease. It is estimated that between 2000 and 2016 53 million lives were saved thanks to the availability of tuberculosis diagnosis and treatment services, which demonstrates the importance of strengthening these research areas.
  • a doctor suspects a patient to be affected by tuberculosis in addition to performing the physical clinical examination, he or she may order the following diagnostic tests: Bronchoscopy, CT scan of the chest, Chest X-ray, clinical laboratory tests (such as ELISA and PCR) , Blood test for gamma interferon secretion (gold standard to check past or active tuberculosis infection), Sputum examination and cultures (bacteriological), Thoracentesis, Tuberculin skin examination, and Biopsy of affected tissue (rare). Radiography and clinical examination are not very specific, since the symptoms of tuberculosis in humans are very similar to those of other diseases, making it necessary to combine more than one examination.
  • the ELISA test is not a cheap test and depending on the complexity of the study, it requires reagents with high costs, therefore, they are developed only in highly specialized medical centers.
  • the tuberculin reaction is a good diagnostic option, but the chances of false positives are high when patients have previously been vaccinated against tuberculosis.
  • bacteriology it is the main diagnostic approach for tuberculosis where the bacterium Mycobacterium tuberculosis is isolated, which can be by two types: smear microscopy (phlegm material is collected and an analysis is made of the presence of M. tuberculosis by microscope visualization) or by simple culture of phlegm material or fluids collected from the patient.
  • smear microscopy phlegm material is collected and an analysis is made of the presence of M. tuberculosis by microscope visualization
  • simple culture of phlegm material or fluids collected from the patient can be by two types: smear microscopy (phlegm material is collected and an analysis is made of the presence of M. tuberculosis by microscope visualization) or by simple culture of phlegm material or fluids collected from the patient.
  • Both approaches correspond to cheap and simple techniques, but a limitation in both techniques is that the patient must already present symptoms, they are hardly performed in patients only carrying the bacteria, being practically impossible to make a subclinical diagnosis of the disease.
  • the bacteria can being in its latent state in the patient for months without developing the symptoms of the disease, becoming a vehicle for the spread of the disease.
  • TB diagnoses are performed in generally through basic pathological tests, and when they need molecular support, the samples are referred to other health institutions, being generally very expensive.
  • US 12 / 594,806 "Immunogenic compositions comprising Mycobacterium tuberculosis polypeptides and fusions thereof" by Reed; Steven G. et al. addresses the detection of the presence of M. tuberculosis in biological samples by contacting the sample with a monoclonal antibody that acts in response to the presence of the antigen (product of the Rv0577 gene), thus detecting the presence of the bacteria.
  • a monoclonal antibody that acts in response to the presence of the antigen (product of the Rv0577 gene), thus detecting the presence of the bacteria.
  • the ABBOTT REALTIME MTB RIF / INH RESISTANCE allows, in a single test, the qualitative detection of resistance for the two most important first-line drugs with activity against M. tuberculosis. It occupies qPCR and its tests can be carried out with material obtained from sputum or fluids, but as was mentioned for the test described previously, although the ABBOTT REALTIME MTB RIF / INH RESISTANCE can be very fast and very sensitive, it is not a test cheap and requires a large amount of sample (sputum or fluids), in addition to being generally occupied when the patient has already developed symptoms.
  • M. tuberculosis detection test from Norten Biotek Corp. This kit works by means of a conventional PCR detecting regions specific to the M. tuberculosis genome. However, according to the manufacturer's specifications, it is indicated that the test can be used only for research purposes, not being recommended for the diagnosis of M. tuberculosis. DESCRIPTION OF THE INVENTION
  • PCR The PCR technique is shown to be the most reliable, inexpensive and simple for the detection of different strains of Mycobacterium tuberculosis. Therefore, in the development of the present invention, PCR was identified as the optimal diagnostic test option, because it solves all the problems identified in the aforementioned techniques, such as: it is a fast technique; it is very specific for detecting different strains of Mycobacterium tuberculosis; It can be applied in samples of patients carrying the bacteria who have not yet developed symptoms (subclinical stage), making it a useful tool for prevention and control; it can be applicable to more than one type of sample; it is cheap, it can be offered to the population in an accessible way; it can be developed in small health centers and hospitals; low false positive rate.
  • the method of the present invention is based on specific and unpublished primers created to identify genes of different strains of Mycobacterium tuberculosis by means of the conventional PCR technique that meet the requirements for the development of a rapid, efficient, sensitive molecular diagnostic test. specific, and low cost.
  • the genes chosen are specific to detect the presence of species of the Mycobacterium tuberculosis Complex made up of the species M. tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microti, which are the main causes of tuberculosis in humans. For this reason, the test will be specific for the strains of Mycobacterium tuberculosis and also broad spectrum, since it allows to identify other species that also cause tuberculosis in mammals, such as those mentioned above.
  • ⁇ X Increase in the number of healthy and diseased samples to improve the technique.
  • x Development of a commercial diagnostic kit.
  • the use of these genes will make it possible to detect the presence of species of the Mycobacterium tuberculosis Complex made up of the species M. tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microti, which are the cause of tuberculosis in humans. .
  • Table 1 Mycobacterium tuberculosis genes chosen for use in PCR assays for diagnosis in human tissue samples according to the present invention.
  • the inventors of the present invention searched the NCBI nr / nt (non-redundant nucleotide) database for the sequences of the four aforementioned genes already described for Mycobacterium tuberculosis. With the selected sequences, the primers were created using the chosen sequences as a guide. The creation of the primers was done in the Primer-BLAST free software from The National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). The advantage of creating primers in this way is due to the possibility of creating new primers based on the basic characteristics that a good primer must possess.
  • melting temperature (T m i) of the primers about 58 "C;
  • the size of the generated amplicon was between 100-300bp;
  • samples correspond to biopsies fixed in paraffin the method according to the present invention admits samples fixed in formalin or in paraffin or, non-fixed samples, of different origin, among which are: lung tissue, cervical lymph nodes , saliva, pleural fluid, bronchoalveolar lavage, sputum, peripheral blood, serum, contaminated milk and biopsies from other mammals, etc.
  • DNA was extracted from the diseased and healthy samples. The material was collected in two tubes per sample. The quantification was carried out in nanodrop and the confirmation of the integrity of the DNA molecule was verified in agarose gel.
  • the diseased samples analyzed were 12 paraffin biopsies of human tissue with probable infection by M. tuberculosis in affected lung tissue and cervical ganglia. These samples were considered as true positive for the reactions according to the present invention. In addition, the samples were confirmed as positive by histopathological analysis.
  • the healthy samples were 6 fresh samples of human oral mucosa without probable infection by M. tuberculosis. Such samples were considered true negative for reactions according to the present invention.
  • PCR assays were carried out with the endogenous human beta-globin gene to certify that the samples were obtained from human tissues and also to certify the integrity of the extracted DNA, to later carry out the assays with the primers of the genes of interest.
  • a DNA concentration gradient was made, the chosen concentration of which was 50ng / ⁇ L for the diseased samples and around 10 ng / ⁇ L for the healthy samples.
  • the concentration used for healthy samples is lower because the material was obtained from a fresh sample, presented high purity detected by NanoDrop, was not subjected to the fixation procedure with formalin (which degrades part of the DNA) and was positive in its amplification in the beta-globin assay, which precisely detects the viability of the nucleic acid in use.
  • Table 3 shows the standard PCR standard reaction to perform the assays.
  • the PCR cycles performed for the beta-globin program and genes of interest correspond to 35 cycles according to the following, changing only the annealing temperature: o 3 minutes at 95 “C; o 30 seconds at 95 “C; or 30 seconds at 53 “C; or 30 seconds at 72"C; I 10 minutes at 72 "C.
  • the PCR reactions were carried out according to what is described in Table 3.
  • the 2% agarose gel electrophoresis of the PCR product was done using the HSP65 pair of primers in diseased and healthy samples. A 100bp molecular weight marker, one blank lane, and two empty lanes were used. The corresponding amplicon is 135 bp.
  • the reactions with the primers to amplify the HSP65 gene indicate that the detection of true positive samples present results with a sensitivity higher than 90%, whose amplifications were visible for almost all the diseased samples, presenting almost 10% of false negative samples, for which is a sensitive technique to detect the presence of M. tuberculosis.
  • the reactions with the primers to amplify the HSP65 gene indicate that the detection of true negative samples presented a 100% effective result, where no amplifications were visualized in any healthy sample, that is, no false positives were obtained.
  • the technique for the HSP65 gene is a specific technique to detect the presence of M. tuberculosis, with a specificity greater than 95% being able to be obtained.
  • the 2% agarose gel electrophoresis of the PCR product was done using the pair of primers Rv0577 in diseased and healthy samples. A 100bp molecular weight marker and blank lane were used. The corresponding amplicon is 125 bp.
  • the same test was repeated for six healthy samples.
  • the reactions with the primers to amplify the Rv0577 gene indicate that the detection of true negative samples presented a result greater than 90%, where no amplifications were visualized in any healthy sample. That is, no false positives were obtained.
  • the technique, in the assay to amplify the Rv0577 gene proved to be a technique with a specificity greater than 95% for detecting the presence of M. tuberculosis.
  • 2% agarose gel electrophoresis of the PCR product was done using the primer pair Rvl513 in diseased and healthy samples. A 100bp molecular weight marker and blank lane were used. The corresponding amplicon is 156 bp.
  • the reactions with the primers to amplify the Rv1513 gene indicate that the detection of true positive samples presents a result with an effectiveness of the order of 80% or higher, presenting false negatives in a range of up to 20% in the samples. Therefore, it is a technique with a sensitivity greater than 80% to detect the presence of M. tuberculosis.
  • the reactions with the primers to amplify the Rv1513 gene indicate that the detection of true negative samples presented a 100% effective result, where no amplifications were visualized in any healthy sample, that is, no false positives were obtained.
  • the Rvl513 amplification assay technique is a technique that achieves a specificity greater than 95% for detecting the presence of M. tuberculosis. PCR reactions for the 143bp expected band gene
  • 2% agarose gel electrophoresis of the PCR product was done using the oxyR pair of primers in diseased and healthy samples. A 50bp molecular weight marker and blank lane were used. The corresponding amplicon is 143 bp.
  • the same test was repeated for six healthy samples.
  • the reactions with the primers to amplify the oxyR gene indicate that the detection of true negative samples presented a result with an effectiveness of at least 83%, where an amplification was visualized in all healthy samples. In addition, a value of 17% false positives was obtained.
  • the technique for the assay to amplify oxyR, on average, is at least 80% specific for detecting the presence of M. tuberculosis.
  • the method for the in vitro detection of Mycobacterium tuberculosis in a sample obtained non-invasively from a patient, is characterized in that it comprises the following steps: a. extract and quantify DNA from the sample; b. perform the Polymerase Chain Reaction using primers for the Rv0577, Rv1513, HSP65 and oxyR genes, c. determine if at least one of the genes achieves positive amplification; and d. assigning the sample as positive for Mycobacterium tuberculosis when it is truly positive, evaluating, for example, the presence or absence of the corresponding amplicon in agarose gel; or, in particular cases, confirming by histopathological analysis.
  • the present invention provides a diagnostic kit for Mycobacterium tuberculosis comprising:
  • Taq polymerase enzyme (5U / ⁇ L), 1 polypropylene tube of 250 ⁇ L;
  • reagents necessary to perform the diagnosis are described below, which although not necessarily part of this kit, are part of those used in routine laboratory techniques in the analysis of the selected sample types, such as extraction and quantification; to determine if there was amplification; and to confirm if there was a positive sample on agarose gel: II.
  • Running buffer TAE IX (40 mM Tris, 20 mM acetic acid, 1 mM EDTA)
  • DNAs were extracted from diseased samples by the modified protocol of Mancilla et al., 2014. Healthy samples were extracted with the commercial E.Z.N.A® Tissue DNA Kit following the manufacturer's recommendations (Omega Bio-TeK).
  • the quality of the DNA samples obtained was evaluated on a 2% agarose gel, run in electrophoresis (90V). To put the DNA samples on the gel, 5 ⁇ L of each sample per well and 5 ⁇ L of running buffer were used.
  • Healthy samples are loaded onto a first gel.
  • the diseased samples are loaded onto a second gel.
  • the ThermoScientific 1Kb Ladder Plus marker was used as molecular weight marker and 2% agarose gel electrophoresis was performed.
  • PCR tests were carried out with the endogenous human beta-globin gene to certify that the samples are human tissues and, in addition, to certify if they are suitable to be used in PCR tests (without degradation and / or inhibitors). This control is necessary before the start of trials with the genes of interest.
  • a DNA concentration gradient was made, the chosen concentration of which was 50ng / ⁇ L for the diseased samples and around 10ng / ⁇ L for the diseased samples. The test was carried out according to the conditions presented in Table 3.
  • the presence or absence of the amplicon (110bp) was evaluated in 2% agarose gel, run in electrophoresis (90V). To put the DNA samples on the gel, 5 ⁇ L of each sample per well and 5 ⁇ L of running buffer were used.
  • Samples were loaded onto two gels. Healthy samples are loaded onto a gel. Diseased samples were loaded onto another gel.
  • the ThermoScientific 1Kb, 50bp and 100bp Ladder Plus marker was used as molecular weight marker.
  • the expected size of the amplified product for beta-globin is approximately 11Opb and it was visualized in 62% of the diseased samples and 100% of the healthy samples, as shown in Table 6.
  • PCR assays were performed with the chosen HSP65 gene to identify the presence of M. tuberculosis in human tissue samples. A concentration gradient of MgCl 2 was made , the chosen concentration of which was 2.5mM. The trial followed what was presented in Table 3.
  • the presence or absence of the amplicon (135bp) was evaluated in 2% agarose gel, run in electrophoresis (90V). To put the DNA samples on the gel, 5 ⁇ L of each sample per well and 5 ⁇ L of running buffer were used.
  • Healthy samples are loaded onto a gel.
  • the diseased samples are loaded onto a second gel.
  • the 100bp ThermoScientific marker was used as a molecular weight marker.
  • the expected size of the amplified product for HSP65 is approximately 135bp and was visualized in 11 of the diseased samples and no healthy samples (Table 7). That pair of primers was 91.7% sensitive and 100% specific for that assay with that number of samples.
  • the presence or absence of the amplicon (143bp) was evaluated in 2% agarose gel, run in electrophoresis (90V). To put the DNA samples on the gel, 5 ⁇ L of each sample per well and 5 ⁇ L of running buffer were used.
  • a concentration gradient of MgCl 2 was made , the chosen concentration of which was 2.5 mM.
  • the healthy samples are loaded onto one gel and the diseased ones are loaded onto the other gel.
  • the 100bp ThermoScientific marker was used as a molecular weight marker.
  • the expected size of the amplified product for oxyR is approximately 143bp and was visualized in 9 of the diseased samples and in 1 of the healthy samples (see Table 8). That pair of primers was 75% specific and 83% sensitive for that assay with that number of samples.
  • the presence or absence of the amplicon (125bp) was evaluated in 2% agarose gel, run in electrophoresis (90V). To put the DNA samples on the gel, 5 ⁇ L of each sample per well and 5 ⁇ L of running buffer were used.
  • a concentration gradient of MgCl 2 was made , the chosen concentration of which was 2.5 mM. Healthy samples are loaded onto one gel, diseased samples are loaded onto the other gel.
  • the 100bp ThermoScientific marker was used as a molecular weight marker.
  • the expected size of the amplified product for Rv0577 is approximately 125 bp and it was visualized in the 12 diseased samples and in no healthy samples. Said pair of primers was 100% specific and 100% sensitive for this test (Table 9) with that number of samples.
  • the presence or absence of the amplicon (156bp) was evaluated in 2% agarose gel, run in electrophoresis (90V). To put the DNA samples on the gel, 5 ⁇ L of each sample per well and 5 ⁇ L of running buffer were used.
  • a concentration gradient of MgCl 2 was made , the chosen concentration of which was 10 mM. All healthy samples are loaded onto one gel and all diseased samples are loaded onto the other gel. The 100bp ThermoScientific marker was used as a molecular weight marker. The expected size of the amplified product for Rv1513 is approximately 156bp and it was visualized in 10 of the 12 diseased samples and no amplicons were seen in the healthy samples (there were no false negatives), see Table 10. This pair of primers is then 100% specific and 83% sensitive for that assay with that number of samples.

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Abstract

La présente invention concerne le domaine du diagnostic clinique et fournit un procédé de diagnostic in vitro pour la détection de différentes souches de Mycobacterium tuberculosis dans des échantillons obtenus à partir de tissus humains par PCR utilisant un ensemble d'amorces ou d'initiateurs spécifiques caractérisé en ce qu'il consiste en un test de diagnostic moléculaire sensible et spécifique. Un autre mode réalisation de la présente invention concerne un système et/ou un kit de diagnostic associé à ce procédé qui utilise ces amorces spécifiques.
PCT/CL2020/050034 2020-04-01 2020-04-01 Procédé et système de diagnostic moléculaire pour la détection de différentes souches de mycobacterium tuberculosis WO2021195794A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2354234A1 (fr) * 1998-12-11 2000-06-22 Visible Genetics Inc. Procede et necessaire pour la caracterisation de mutations produites par une resistance aux antibiotiques chez le mycobacterium tuberculosis
WO2018012896A1 (fr) * 2016-07-13 2018-01-18 주식회사 큐라티스 Ensemble d'amorces pcr multiplex pour le diagnostic simultané de mycobacterium tuberculosis ou de mycobactéries non tuberculeuses, ainsi que composition et kit comprenant cet ensemble

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2354234A1 (fr) * 1998-12-11 2000-06-22 Visible Genetics Inc. Procede et necessaire pour la caracterisation de mutations produites par une resistance aux antibiotiques chez le mycobacterium tuberculosis
WO2018012896A1 (fr) * 2016-07-13 2018-01-18 주식회사 큐라티스 Ensemble d'amorces pcr multiplex pour le diagnostic simultané de mycobacterium tuberculosis ou de mycobactéries non tuberculeuses, ainsi que composition et kit comprenant cet ensemble

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Title
DVORSKÁ L., BARTOŠ M., MARTIN G., ERLER W., PAVLÍK I.: "Strategies for differentiation, identification and typing of medically important species of mycobacteria by molecular methods", VETERINARNI MEDICINA, CESKA AKADEMIE ZEMEDELSKYCH VED,CZECH ACADEMY OF AGRICULTURAL SCIENCES, CZ, vol. 46, no. No. 11–12, 1 January 2001 (2001-01-01), CZ , pages 309 - 328, XP055935823, ISSN: 0375-8427, DOI: 10.17221/7890-VETMED *
GUANGYU XU, YUE SHI, XIAOYU SUN , CHENGGUO WEI , GUOQING WANG, FAN LI: "Identification and Validation Of Highly Specific And Stable Diagnostic Targets In Mycobacterium tuberculosis", JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, 9 November 2015 (2015-11-09), pages 9 - 14, XP055935820, Retrieved from the Internet <URL:https://oaji.net/articles/2016/2409-1455876123.pdf> [retrieved on 20220627] *
HUARD RICHARD C., DE OLIVEIRA LAZZARINI LUIZ CLAUDIO, BUTLER W. RAY, VAN SOOLINGEN DICK, HO JOHN L.: "PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 41, no. 4, 1 April 2003 (2003-04-01), US , pages 1637 - 1650, XP055935819, ISSN: 0095-1137, DOI: 10.1128/JCM.41.4.1637-1650.2003 *
MUKHERJEE F., BAHEKAR VS, SUBRAMANIAN BM, PASHA SY, PRASAD A, SURENDRA KSNL, RANA SK, SHARMA GK, PREMALATHA D, SRINIVASAN VA: "Real-Time PCRs that Discriminates Mycobacterium tuberculosis and Mycobacterium bovis Based on Single Nucleotide Polymorphism and Genome Analysis of Amplicons", ADVANCES IN ANIMAL AND VETERINARY SCIENCES, vol. 7, no. 1, 1 January 2018 (2018-01-01), XP055935821, ISSN: 2307-8316, DOI: 10.17582/journal.aavs/2019/7.2.49.65 *

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