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WO2017077999A1 - Procédé de détection d'adn dérivé du complexe tuberculosis - Google Patents

Procédé de détection d'adn dérivé du complexe tuberculosis Download PDF

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WO2017077999A1
WO2017077999A1 PCT/JP2016/082390 JP2016082390W WO2017077999A1 WO 2017077999 A1 WO2017077999 A1 WO 2017077999A1 JP 2016082390 W JP2016082390 W JP 2016082390W WO 2017077999 A1 WO2017077999 A1 WO 2017077999A1
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dna
primer
probe
mycobacterium tuberculosis
seq
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昌樹 山本
良太 牛尾
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Yokohama City University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a primer and probe set for detecting DNA derived from Mycobacterium tuberculosis by digital PCR (hereinafter sometimes referred to as “dPCR”), and DNA derived from Mycobacterium tuberculosis from a blood sample using the set. It relates to the detection method.
  • dPCR digital PCR
  • Non-patent Document 1 Pulmonary tuberculosis is still one of the threatening infectious diseases, and 9 million people develop and 1.5 million people die every year worldwide (Non-patent Document 1). As a diagnostic method for pulmonary tuberculosis, sputum smear and culture tests are the gold standard. However, this test method often has difficulty in diagnosis, and delays in diagnosis may cause problems such as spread of infection and induction of tuberculosis resistance.
  • Non-Patent Documents 2 to 8 Nucleic acid amplification testing methods such as PCR and LAMP have been developed as diagnostic methods to compensate for the drawbacks.
  • a repetitive sequence IS6110, a gyrase subunit B (gyrB) gene, and the like are used as target regions for specifically detecting Mycobacterium tuberculosis (Patent Documents 1 and 2, etc.).
  • specimens usually use sputum in the nucleic acid amplification test method. When collecting sputum samples, careful precautions are required to prevent the spread of pathogens. Sometimes it is difficult to collect sputum, and diagnosis is often difficult. Moreover, since tuberculosis bacteria infect other than respiratory organs, invasive treatment may be necessary to obtain a suitable specimen.
  • Non-Patent Documents 9 to 14 Nucleic acid amplification test methods using urine, peripheral blood mononuclear cells, etc. have been reported as a sample that replaces the minimally invasive sputum. However, these examination methods have not yet been clinically used in terms of sensitivity.
  • An object of the present invention is to provide means capable of accurately and promptly diagnosing Mycobacterium tuberculosis infection using a sample that is minimally invasive, has a low risk of infection spread, and can be collected stably.
  • M. tuberculosis DNA Due to the fact that M. tuberculosis DNA is detected in the urine, the present inventors may have a very low concentration of cell-free free M. tuberculosis DNA in the plasma of patients infected with M. tuberculosis. I thought that there was sex. Focusing on the recently developed digital PCR technology that can demonstrate high sensitivity in detecting extremely low copy number DNA, we aim to establish a method to detect M. tuberculosis-derived DNA in plasma by digital PCR. As a result of diligent research on the design of primers and probes capable of detecting various strains of, and the conditions of digital PCR, plasma of tuberculosis patients was obtained by digital PCR using specific primer and probe sets targeting IS6110 and gyrB. The present invention was completed by successfully detecting M. tuberculosis group-derived DNA at a level of several copies / 20 ⁇ L reaction solution.
  • the present invention is a digital PCR primer for detecting Mycobacterium tuberculosis group IS6110, comprising a forward primer having the base sequence shown in SEQ ID NO: 1, a reverse primer having the base sequence shown in SEQ ID NO: 2, and a probe having the base sequence shown in SEQ ID NO: 3. And a probe set.
  • the present invention also relates to a digital PCR primer for detecting Mycobacterium tuberculosis group gyrB, comprising a forward primer having the base sequence shown in SEQ ID NO: 4, a reverse primer having the base sequence shown in SEQ ID NO: 5, and a probe having the base sequence shown in SEQ ID NO: 6. And a probe set.
  • the present invention comprises a tuberculosis group comprising DNA PCR extracted from a blood sample isolated from a subject and performing digital PCR using at least one of the primer and probe set of the present invention.
  • a method for detecting a derived DNA is provided.
  • the present invention provides a tuberculosis group-derived DNA detection reagent or kit for digital PCR, comprising at least one of the primer and probe set of the present invention.
  • a means for specifically detecting a tuberculosis group-derived DNA by distinguishing it from non-tuberculous mycobacteria from a plasma sample to enable detection of M. tuberculosis group infection and the like is provided.
  • M. tuberculosis group-derived DNA can be detected even with a low copy number of less than 10 copies per reaction solution, and the detection sensitivity is extremely high.
  • Blood samples can be obtained stably, and samples can be obtained from subjects such as children who are difficult to collect respiratory-derived samples such as sputum and endoscope washing liquid. Even in cases infected with respiratory organs, blood samples can be collected with minimal invasiveness.
  • a minute amount of DNA derived from Mycobacterium tuberculosis group contained in the plasma of a patient infected with Mycobacterium tuberculosis group is detected by digital PCR.
  • Digital PCR is a technique in which a PCR reaction solution is divided into a large number of small compartments, PCR is performed, and target DNA is detected and quantified based on the number of small compartments in which amplification is detected.
  • tuberculosis group includes Mycobaterium tuberculosis (MTB), M. bovis, M. ⁇ africanum, M. microti, M. canettii, M. caprae, and M. pinnipedii. Of these, the one that causes tuberculosis in humans is primarily M. tuberculosis. Since the gene region targeted in the present invention is conserved in the above-mentioned Mycobacterium belonging to the Mycobacterium group, various strains of the Mycobacterium group can be detected. In the present invention, the term “detection” includes qualitative detection, quantitative detection, and semi-quantitative detection.
  • One of the digital PCR primers and probe sets for detecting Mycobacterium tuberculosis is targeted at the repetitive sequence IS6110 (GenBank accession number X17348 etc.) present in the Mycobacterium tuberculosis complex genome.
  • the nucleotide sequences of the forward primer, reverse primer, and probe for detecting IS6110 are shown in SEQ ID NOs: 1 to 3, respectively.
  • This set for detecting IS6110 targets the region from position 311 to position 381 in the base sequence shown in SEQ ID NO: 11.
  • SEQ ID NO: 11 is a part of the IS6110 sequence of Mycobacterium tuberculosis M. tuberculosis, and is a region adopted as a DNA standard for IS6110 in the following examples.
  • Another digital PCR primer and probe set for detecting Mycobacterium tuberculosis group targets genes encoding gyrase subunit B (gyrB, GenBank accession number AL123456.3, etc.).
  • the base sequences of gyrB detection forward primer, reverse primer and probe are shown in SEQ ID NOs: 4 to 6, respectively.
  • This set for detecting gyrB targets the region at positions 217 to 334 in the base sequence shown in SEQ ID NO: 12.
  • SEQ ID NO: 12 is a part of the gyrB sequence of Mycobacterium tuberculosis M. tuberculosis and is a region adopted as a DNA standard for gyrB in the following examples.
  • the dPCR probe can basically adopt the same principle as the real-time PCR probe.
  • the dPCR probe for detecting IS6110 and the dPCR probe for detecting gyrB may detect amplification by any of the intercalator method, TaqMan (registered trademark) probe method, and cycling probe method, but TaqMan (registered trademark) )
  • the probe method can be preferably employed.
  • a fluorescent dye as a reporter substance is bound to one end of the probe, and a quencher substance is bound to the other end.
  • a reporter substance is bound to the 5 ′ end and a quencher substance is bound to the 3 ′ end.
  • Various fluorescent dyes and quencher substances are known.
  • any fluorescent dye and quencher substance can be used for the dPCR probe for IS6110 detection and the dPCR probe for gyrB detection as long as the quenching of fluorescence before probe decomposition and the generation of fluorescence after probe decomposition occur appropriately. May be.
  • a PCR reaction solution is divided and injected into a very small reaction well.
  • the technique of performing is known. Any digital PCR may be used in the present invention.
  • the former method is called a droplet method, and the latter method is called a microwell method.
  • droplets formed in oil correspond to “small compartments”
  • reaction wells into which PCR reaction solutions are dividedly injected correspond to “small compartments”.
  • a very small amount of free (cell-free) Mycobacterium tuberculosis group-derived DNA present in the blood of a subject infected with M. tuberculosis group It can be detected with sensitivity.
  • the Mycobacterium spp. That cause diseases by infecting mammals such as humans include a group of bacteria collectively called non-tuberculous mycobacteria in addition to the Mycobacterium tuberculosis group. According to dPCR, it is possible to specifically detect the Mycobacterium tuberculosis group in distinction from nontuberculous mycobacteria.
  • each step of the method for detecting DNA derived from Mycobacterium tuberculosis by dPCR using the primer and probe set of the present invention will be described. It should be noted that at least one of IS6110 detection primer and probe set and gyrB detection primer and probe set may be used for detection of DNA derived from Mycobacterium tuberculosis group. If each assay is performed using two sets for one specimen, the accuracy of detection of Mycobacterium tuberculosis group can be further increased.
  • DNA used as a dPCR sample is prepared from a blood sample isolated from a subject.
  • the subject is a mammal, typically a human.
  • the term blood sample includes whole blood, plasma, and serum.
  • DNA may be extracted from serum, it is convenient and preferable to separate plasma from a whole blood sample and extract DNA from plasma to obtain a dPCR sample. Extraction of plasma DNA can be easily performed using a commercially available kit or the like. Methods for detecting M. tuberculosis-specific DNA in peripheral blood mononuclear cells are known (Condos R, et al. Lancet 1996, 347 (9008), p.1082-5 .; Taci N, et al.
  • the dPCR reaction solution can be prepared using a commercially available dPCR reagent containing a heat-resistant polymerase.
  • the primer concentration is preferably about 700 nM to 1100 nM, for example, about 800 nM to 1000 nM, and the probe concentration is about 100 nM to 150 nM, for example, 110 nM to 135 nM. . If the concentration of the primer is lower than this, the intensity of the fluorescence signal from each small section after the amplification reaction is greatly reduced, which is not desirable.
  • the probe concentration is higher than this, it is easy to generate false positives or gray zone subdivisions (called rain drops in the droplet method) that show intermediate fluorescence intensities that are difficult to determine as either positive or negative. Is not desirable.
  • the amount of the subject DNA sample when the total amount of the reaction solution is 20 ⁇ L, DNA corresponding to about 20 ⁇ L of plasma may be used.
  • the dPCR reaction solution is divided into a large number of small compartments.
  • the dPCR reaction solution is divided into a large number of small droplets in oil to form an emulsion.
  • the dPCR reaction solution is dividedly injected into a microreaction well on the chip.
  • division into small sections can be performed using a commercially available dedicated device.
  • the dPCR reaction solution may be divided so that the target DNA has a concentration of about 1 to 2 copies or less in a small compartment, and is not particularly limited, but the PCR reaction solution containing the subject DNA sample with the above total DNA amount What is necessary is just to divide 20 microliters into about tens of thousands, for example, about 10,000 to 30,000 small sections.
  • the amplification reaction After dividing into small sections, perform an amplification reaction using an appropriate PCR device.
  • the number of samples that can be processed simultaneously in a single run of an amplification reaction varies depending on the dPCR system used, but it is usually preferable to perform the amplification reaction immediately after the division of the dPCR reaction solution (particularly in the case of the droplet method). Divide the dPCR reaction solution as much as can be processed in one run.
  • the temperature of annealing and extension reaction it is desirable that the temperature of annealing and extension reaction be about 52 ° C. to 55 ° C. in both the IS6110 detection system and the gyrB detection system. When an annealing and extension reaction is performed at this temperature, a high fluorescence signal can be obtained after the amplification reaction.
  • the fluorescence signal from each small compartment is detected with an appropriate fluorescence reader.
  • a commercially available fluorescence reader dedicated to the dPCR system used may be used. Since the fluorescence signal is generated in the small compartment where the amplification has occurred, the number is counted with the small compartment producing the fluorescence as the positive small compartment.
  • the tuberculosis group-derived DNA is present in the blood of the subject, Therefore, it can be determined that the subject is infected with the Mycobacterium tuberculosis group.
  • the method of the present invention detects the infection of Mycobacterium tuberculosis group. In addition, it can also be used for monitoring tuberculosis group infection or the disease state of tuberculosis, evaluating the risk of developing tuberculosis, and determining the therapeutic effect of tuberculosis group infection or tuberculosis. That is, the method of the present invention can be performed to assist these actions by the physician.
  • the threshold can be set by dPCR using an appropriate DNA standard as a template.
  • a target gene region partial fragment containing the amplified region of the dPCR primer may be used.
  • a DNA standard for the IS6110 detection system a DNA fragment having the base sequence shown in SEQ ID NO: 11 is used, and as a DNA standard for the gryB detection system, a DNA fragment having the base sequence shown in SEQ ID NO: 12 is used. be able to.
  • Such a DNA fragment can be obtained by amplification by PCR using the genomic DNA of the Mycobacterium tuberculosis group (for example, M.
  • tubatuberculosis standard strain as a template.
  • the DNA standard is used as a template in the form of a linear DNA fragment, not in the form of circular DNA incorporated into plasmid DNA. By using it in a straight chain, the occurrence of gray zone small sections called rain drops in the droplet method can be greatly reduced, and an appropriate threshold value can be set.
  • the DNA standard reaction may be performed at least once per run of amplification reaction.
  • the dPCR reaction solution after dividing into small compartments is transferred to a 24-well plate or a 96-well plate and an amplification reaction is performed, it is sufficient to provide at least one well of the DNA standard reaction per plate.
  • at least one DNA standard microwell chip may be added to one run.
  • the fluorescence signal of each subcompartment is detected by the fluorescence reader in the same way as the subject sample, and the positive and negative subcompartments are appropriately determined by cluster analysis of the signal intensity of each subcompartment.
  • the threshold value to be divided is determined.
  • the cluster analysis is preferably performed by the nearest neighbor method. For example, the known k-nearest neighbor algorithm “definetherain” (Jones M, et al. J Virol Methods 2014; 202: 46-53.) Is preferably used. Can do.
  • the determined threshold value is applied to the subject sample that has been amplified simultaneously with the DNA standard. For example, when the amplification reaction of a large number of subject-derived samples is performed twice on two plates, the DNA standard added to the same first plate for the sample on the first plate Apply the threshold determined from the dPCR result of the sample, and apply the threshold determined from the dPCR result of the DNA standard added to the second plate to the second plate sample. Judgment of positive / negative of a small section of a sample derived from a person. A small compartment with a signal intensity exceeding the threshold is judged positive. In order to check for false positives, dPCR may also be performed as a negative control for the reaction solution without addition of template DNA.
  • the above-mentioned primers and probe sets for detecting DNA derived from Mycobacterium tuberculosis can be provided as a DNA detection reagent or kit for Mycobacterium tuberculosis for dPCR. Only one of the two types of primers and probe sets may be included, or both may be included.
  • the primer and probe may be in a dried form, in a form dissolved in a buffer solution or the like, or in a form in which the dried primer and probe are combined with a buffer solution or the like.
  • the reagent or kit may be in the form of a set of the linear DNA standards described above. Furthermore, the reagent or kit may be in the form of a set of other reagents necessary for dPCR.
  • Reagents other than primers and probes necessary for dPCR are well known.
  • a buffer solution for preparing a dPCR reaction solution, a heat resistant polymerase, or a dPCR containing a heat resistant polymerase and dNTPs in the buffer solution examples include a premix solution for preparing a reaction solution. Instructions for use are usually attached to the reagent or kit.
  • Pulmonary tuberculosis (PTB) patients were patients who were diagnosed with sputum smear positive pulmonary tuberculosis and were isolated and treated at Yokohama City University Hospital. PTB was diagnosed by a conventional nucleic acid amplification test using sputum specimens that were positive for acid-fast bacteria. Healthy controls were selected from the health care workers at the hospital. Regarding healthy controls, it was confirmed that there was no MTB infection history by a commercially available ELISPOT assay (T-SPOT.TB).
  • Non-tuberculous mycobacteria (NTM) patients were patients who met the diagnostic criteria of the American Thoracic Society and the American Infectious Diseases Society (Gutierrez-Aguirre I, et al. Methods Mol Biol 2015, 1302, p. 331-47.). Eleven strains of NTM obtained from sputum or bronchial lavage fluid of NTM patients (M. abscessus, M. avium, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. marinum, M. scrofulaceum, M. szulgai, M. terrae).
  • Sample preparation Peripheral blood was collected from each subject using a vacutainer blood collection tube (Terumo) containing ETDA-2Na. Whole blood was centrifuged at 1500 rpm for 10 minutes to obtain a plasma sample. Total DNA was extracted from 200 ⁇ L of each plasma sample using Qiagen DNeasy Blood and Tissue Kit (Qiagen). The kit was basically used according to the attached instructions except that the amount of Buffer AE used during elution was reduced to 40 ⁇ L. All samples were processed within 8 hours after collection. Genomic DNA of each NTM strain was extracted from colonies on an egg-based solid medium (Ogawa medium) using the Qiagen DNeasy Blood and Tissue Kit. The extracted DNA sample was stored at ⁇ 80 ° C. until analysis.
  • Qiagen DNeasy Blood and Tissue Kit Qiagen DNeasy Blood and Tissue Kit
  • dPCR Primers and Probes Two sets of primers and probes were designed for detection of MTB specific genomic DNA sequences. One targeted the insertion sequence 6110 (IS6110; GenBank accession number X17348). This insertion sequence is conserved within the Mycobacterium tuberculosis group including M. tuberculosis (MTB), M. bovis, M. africanum, M. microti, M. canettii, M. caprae, and M. pinnipedii (Thierry D , et al. J Clin Microbiol 1990, 28 (12), p.2668-73.). The base sequences of primers and probes used for amplification of IS6110 are shown below.
  • the probe was bound with 6-carboxyfluorescein (FAM) as a reporter dye at the 5 'end and black hole quencher (BHQ-1) as a quencher dye at the 3' end.
  • FAM 6-carboxyfluorescein
  • BHQ-1 black hole quencher
  • Gyrase subunit B (gyrB; GenBank accession number AL123456.3). gyrB is also conserved within the Mycobacterium tuberculosis group (Kasai H, et al. J Clin Microbiol 2000, 38 (1), p.301-8; Niemann S, et al. J Clin Microbiol 2000, 38 (9), p.3231-4.). The base sequences of primers and probes used for amplification are shown below. The dye was bound to the probe in the same manner as the IS6110 probe.
  • gyrB forward AAGGACCGCAAGCTACTGAA (SEQ ID NO: 4)
  • gyrB reverse GTGTTGCCCAACTTGGTCTT (SEQ ID NO: 5)
  • gyrB probe [FAM] -ACCTCACCGGTGACGATATC- [BHQ-1] (SEQ ID NO: 6)
  • the double-stranded DNA fragment containing the region to be detected is the MTB standard strain (JATA KK11-291) genomic DNA as a template, and the primer set is IS6110 PC-F (AACGGCTGATGACCAAACTC, SEQ ID NO: 7) And IS6110 PC-R (GATCGTCTCGGCTAGTGCAT, SEQ ID NO: 8), or gyrB PC-F (CAAGAACGCGATTCATAGCA, SEQ ID NO: 9) and gyrB PC-R (TGGGTCAGCTGTTCGTTACA, SEQ ID NO: 10).
  • Each fragment was ligated into pCR-Blunt II TOPO vector (Invitrogen) and introduced into E.
  • coli DH5 ⁇ TOYOBO. Recombinant clones were selected on LB agar medium containing kanamycin (KM) and cultured in LB liquid medium containing KM. Plasmids were isolated using HighSpeed Plasmid Midi Kit (Qiagen) according to manufacturer's instructions. The concentration of plasmid DNA was calculated based on the molecular weight of the DNA, and the DNA solution was diluted with Tris-EDTA Buffer (Sigma-Aldrich) to 1.0 ⁇ 10 4 copies / ⁇ L.
  • Tris-EDTA Buffer Sigma-Aldrich
  • dPCR reaction solution for clinical specimen assay was prepared as follows; mix 10 ⁇ L dPCR Probe Supermix (BioRad), 900 nM each primer, 125 nM probe, 4 ⁇ L sample. DNase and RNase-free ultrapure water was used to make 20 ⁇ L.
  • the reaction solution was loaded on a QX200 Droplet Generator (BioRad) to generate microdroplets. The produced emulsion of droplets was transferred to a 96-well PCR plate (Eppendorf), sealed with a foil heat seal (Eppendorf), and heated at 180 ° C. for 5 seconds. Subsequently, amplification reaction was performed using C1000 touch thermal cycler (BioRad). The reaction conditions were 95 ° C.
  • the temperature gradient rate was set to 2.0 ° C./second.
  • the emulsion produced by the Droplet Generator was immediately subjected to an amplification reaction, and the endpoint fluorescence signal from each droplet in the emulsion was measured using a QX200 Droplet Reader (BioRad). Each sample and each target assay was performed in duplicate. To avoid contamination inside the QX200 Droplet Reader, the standard fluorescence was analyzed last.
  • the fluorescence intensity data after amplification reaction of each droplet was exported as a csv file from QuantaSoft droplet reader software (version 1.7.4, BioRad).
  • the threshold for selecting positive droplets depends on the fluorescence intensity of the standard droplets applied to the k-nearest neighbor algorithm “definetherain” (Jones M, et al. J Virol Methods 2014, 202, p.46-53.). Were determined.
  • ⁇ Result> 1 Optimization of dPCR conditions (1) Examination of annealing temperature The dPCR reaction conditions were optimized using DNA standards prepared for each of the two target genes. First, gradient PCR amplification was performed in the range of 50 ° C. to 60 ° C. in order to determine the annealing temperature. As a result, all of the IS6110 detection primer and the gyrB detection primer had Tm values of about 60 ° C., but the fluorescence intensity was highest at 54 ° C. in any assay (FIG. 1). Therefore, the temperature of annealing and extension reaction in dPCR of clinical specimens was set to 54 ° C.
  • FIG. 2 shows the result of dPCR using the DNA standard prepared above as a template at a concentration of 10 4 to 10 9 and an annealing and extension reaction temperature of 54 ° C. Drop was recognized. Since it is not possible to define an appropriate threshold value as it is, measures for this rain drop were examined.
  • the plasmid was cleaved on both sides of the insert by EcoRI digestion, and dPCR was performed using the linearized insert fragment as a template. As a result, as shown in FIG. 3, raindrops were significantly reduced. This makes it possible to accurately define the threshold value.
  • the recommended manufacturer concentration of primers and probes is 900 nM for the primer and 250 nM for the probe.
  • the primer and probe for IS6110 were subjected to dPCR using EcoRI-digested standard DNA as a template and the primer and probe concentrations varied.
  • the primer concentration was 450 nM and 900 nM
  • the probe concentration was 125 nM and 250 nM. The results are shown in FIG. When the primer concentration was 450 nM, the fluorescence intensity was significantly reduced. Therefore, it was considered that the primer should be 900 nM.
  • the probe concentration was compared between 125 nM and 250 nM in both the IS6110 detection system and the gyrB detection system. The results are shown in FIG. At a probe concentration of 250 nM, one false positive droplet was observed in the IS6110 detection system. Also, 250nM had more raindrops than 125nM. The purpose of this study was to detect low copy number of DNA, so the appropriate probe concentration was 125 nM with few false positives.
  • Detectability of plasma DNA in PTB patients was evaluated by receiver operating characteristic (ROC) analysis (FIG. 7).
  • the sensitivity of IS6110 detection is 83% and the specificity is 93% at the DNA concentration of the 1.5 copy / 20 ⁇ L reaction solution, and the sensitivity of gyrB detection is 58% and the specificity at the DNA concentration of the 0.35 copy / 20 ⁇ L reaction solution.
  • ROC receiver operating characteristic
  • the IS6110 detection assay was 36 copies / 20 ⁇ L and the gyrB detection assay was 6.3 copies / 20 ⁇ L.
  • the IS6110 detection assay was over 2000 copies / 20 ⁇ L (2430 and 2260 copies / 20 ⁇ L, respectively). One of the two had very severe clinical symptoms and died 3 weeks after starting standard treatment.
  • NTM strains showed a maximum of 2 positive droplets per assay and were evaluated as false positives as above.
  • the dPCR assay system established here was able to detect cell-free M. tuberculosis group DNA present in minute amounts in the blood of patients infected with M. tuberculosis group. Even less than 10 copies per reaction solution can be quantified, confirming that the detection sensitivity is very high.
  • qPCR quantitative real-time PCR
  • dPCR assay system established by the present inventors using blood samples from 24 patients with sputum smear-positive pulmonary tuberculosis .
  • qPCR uses the same primers and probes used in dPCR, including 10 ⁇ L TaqMan Fast Advanced Master Mix (Applied Biosystems, USA Foster City), 900 nM each primer, 125 nM probe, 4 ⁇ L template DNA The reaction was performed in 20 ⁇ L of the reaction solution. Reaction solutions were prepared in duplicate for each sample, and the reaction was performed in a 96-well reaction plate using StepOnePlus Real-Time PCR System (Applied Biosystems).
  • reaction conditions were 95 ° C. for 20 seconds and 60 ° C. for 20 seconds for 40 cycles.
  • a 10-fold serial dilution series of plasmid DNA standards of IS6110 and gyrB was prepared in duplicate and included in each plate, and these were measured to create a standard curve.
  • the patient was a 63-year-old male who was severely immunocompromised due to hematopoietic stem cell transplantation for the treatment of acute myeloid leukemia. Fever and inflammation continued, with pancytopenia and abnormal clotting.
  • Whole body CT scan revealed many granular shadows throughout the lung, suggesting disseminated MTB infection.
  • anti-acid staining, culture and conventional MTB real-time PCR test Cobas TaqMan MTB, Roche Diagnostics, Basel, Switzerland
  • T-SPOT. TB test (Oxford Immunotec, Oxford UK) are all negative. Met.
  • the urine Mycobacterium culture was positive, but the real-time PCR test (Cobas TaqMan MTB / MAI, Roche Diagnostics) for MTB and MAC (Mycobacterium avium complex) was negative. It was not possible to identify what was cultured. A biopsy of the liver or bone marrow for further examination was infeasible due to bleeding characteristics.
  • circulating MTB-DNA in plasma was detected using the blood sample of the patient by the dPCR assay system targeting the IS6110 gene established by the inventors of the present invention as described above.
  • the conditions for dPCR were such that the primer concentration was 900 nM, the probe concentration was 125 nM, and the annealing and extension reaction temperatures were 54 ° C.
  • the threshold was determined manually, and droplets exceeding the threshold were considered positive.
  • the DNA standard the plasmid DNA containing the IS6110 gene fragment constructed above was digested with EcoRI and linearized. The results of repeated analysis were all positive (16.4 copies / well, 9.8 copies / well), and MTB infection was detected (FIG. 8).

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  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
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Abstract

Suite à une recherche poussée de conceptions d'amorces et de sondes, de conditions de PCR numérique, et analogues avec lesquelles les diverses souches bactériennes du complexe tuberculosis peuvent être détectées afin d'établir un procédé de détection d'ADN dérivé du complexe tuberculosis dans un plasma par PCR numérique, les inventeurs de la présente invention ont établi un procédé grâce auquel un ADN dérivé du complexe tuberculosis peut être détecté à partir du plasma d'un patient tuberculeux sur un niveau de plusieurs copies/20 µl de solution de réaction par PCR numérique au moyen d'une amorce et d'une sonde spécifiques ciblant IS6110 et gyrB.
PCT/JP2016/082390 2015-11-06 2016-11-01 Procédé de détection d'adn dérivé du complexe tuberculosis Ceased WO2017077999A1 (fr)

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CN112725475A (zh) * 2021-01-05 2021-04-30 四川大学华西医院 一种结核分枝杆菌检测引物和探针组合物、试剂盒及应用
US11174520B2 (en) 2018-02-27 2021-11-16 Delta Electronics, Inc. Method for detecting presence or absence of Mycobacterium and kit thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11174520B2 (en) 2018-02-27 2021-11-16 Delta Electronics, Inc. Method for detecting presence or absence of Mycobacterium and kit thereof
JP2020022451A (ja) * 2018-08-08 2020-02-13 台達電子工業股▲ふん▼有限公司Ｄｅｌｔａ Ｅｌｅｃｔｒｏｎｉｃｓ，Ｉｎｃ． マイコバクテリウムの検出方法及びそのキット
CN112725475A (zh) * 2021-01-05 2021-04-30 四川大学华西医院 一种结核分枝杆菌检测引物和探针组合物、试剂盒及应用

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