RU2636458C1 - Method for increase of real time polymerase chain ​​reaction sensitivity in detection of pathogenic bacteria dna - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to laboratory methods of investigation in microbiology, namely, to methods for pathogenic bacteria(Mycobacterium leprae, Mycobacterium tuberculosis complex (MTBC) and Treponema pallidum) DNA detection using real-time polymerase chain reaction. The invention is intended for use in microbiological and genetic laboratories that solve the problems of pathogenic microorganisms DNA detection during the initial diagnosis of bacterial infections, as well as to evaluate the effectiveness of therapeutic measures and cure control.EFFECT: increased the sensitivity of the polymerase chain reaction when pathogenic bacteria DNA is detected using primers and DNA probes to specific repeating elements of their genome present in the bacterial chromosome in an amount of two or more copies, decreased number of amplification cycles necessary to achieve a positive polymerase chain reaction in case of detectable microorganism presence in the analysed sample, positive result of polymerase chain reaction-detection when the use of primers and DNA probes to sequences of genes present in the bacterial chromosome in single copies gives a negative result.4 cl, 10 dwg, 3 ex

Description

The invention relates to medicine, namely to laboratory methods of research in microbiology, and is intended to detect pathogenic bacteria, including Mycobacterium leprae, Mycobacterium tuberculosis complex (MTBC) and Treponema pallidum. In particular, the invention relates to methods for detecting DNA of pathogenic bacteria using nucleic acid amplification methods, namely, real-time polymerase chain reaction. The invention can be used to detect M. leprae, MTBC and T. pallidum in clinical samples during the initial diagnosis of the corresponding bacterial infections, as well as to evaluate the effectiveness of therapeutic measures and control of cure.

The prior art [1 - Mullis K.V., Faloona FA. Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Method Enzymol. 1987, 155, 335] the method of polymerase chain reaction (PCR) is known, the essence of which is that short nucleotide sequences are introduced into the test sample, presumably containing one or another infectious agent — primers (direct and reverse), complementary to specific fragments of the desired genome pathogen. With subsequent heat treatment, denaturation of the double-stranded DNA of the pathogen is achieved, making it available for interaction with primers complementary to the corresponding regions of genomic DNA. At the next stage, in the presence of deoxynucleotide triphosphates and DNA polymerase in the reaction mixture, selective reproduction (amplification) of a DNA region of a certain size occurs, which is set when choosing primers. The cyclic repetition of DNA denaturation and renaturation leads to an exponentially increasing accumulation of specific DNA fragments available for visual registration in agarose or acrylamide gels. Improved versions of this method use thermostable DNA polymerase of the thermophilic microorganism Thermus aquaticus [2 - US 4889818 A, Purified thermostable enzyme], which allows for PCR without the addition of an enzyme in each reaction cycle, as well as fluorescence-labeled probes that allow the measurement of the amount of amplified DNA in real time after each amplification cycle. In particular, the most common detection method - the use of linear destructible samples (TaqMan) is based on the use of a fluorescently-labeled oligonucleotide, complementary to the internal sequence of the amplified DNA fragment of the pathogen, with a fluorescent label in the 5'-position and a fluorescence quencher in the 3'-position [3 - US 20110300544 A1, Enhanced taqman probe based amplification].

Based on this universal principle, many methods have been developed and patented for identifying bacterial infections, the differences between which are in the primary nucleotide sequences of the primers used. In this case, the key requirement is the uniqueness of this sequence: (a) a sequence complementary to it (gene or gene fragment) must be present in the genome of all representatives of the detected bacterial species; (b) similar sequences should not be present in the genomes of other bacterial species. Compliance with these requirements ensures that PCR technologies implemented on their basis comply with the “specificity” requirement.

To identify M. leprae, the use of primers 5'-CGGCTTCACGTCCAGTTTCTTC-3 'and 5'-TAAGTGCCCTCGATGTAAGCGG-3' for a fragment of the MntH gene encoding Mn ²⁺ , a transport protein of this microorganism, was proposed [4 - Cruz AF, Furini RBar Roselino microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy. An Bras Dermatol. 2011 86 (4): 651-6.], Primers 5'-GTGGTCGGCCTCTCGAT-3 'and 5'-CGAGCCAGCATAGATGAACTGATC-3' to a fragment of the fbp gene encoding a fibronectin binding protein [5 - Martinez et al., Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy. J Clin Microbiol. 2006 Sep; 44 (9): 3154-9.], As well as other primers for a number of other species-specific fragments of the genome of this bacterium - 16S rRNA, rpoB, sodA, etc.

Known solutions to the MTBC DNA detection problem are based on the use of primers 5'-GCACCCGCGCCGAGTTAG-3 'and 5'-GGCCAGCCGATCCAATACCC-3' to a fragment of the recX gene encoding a specific regulatory protein [6 - Bhatter et al., Kinetics of recA and recX induction in drug -susceptible and MDR clinical strains of Mycobacterium tuberculosis. J Antimicrob Chemother, doi: 10.1093 / jac / dku319.], 5'-CACGTAGGCG AACCCTGCCC AGGTC-3 ', 5'-GCGTAGGCGTCGGTCACAAA-3' to the unique Mycobacterium tuberculosis DNA sequence [7 - US One, 010160527, PCT1010605 detecting Mycobacterium tuberculosis], as well as primers complementary to fragments of rpoB, katG, gyrB, hupB, etc. genes.

In turn, to solve the T. pallidum detection problem, the use of primers 5'-TGCGCGTGTGCGAATGGTGTGGTC-3 'and 5'-CACAGTGTCCAAAAAACGCCTGCACG-3' to a unique region in the coding DNA polymerase I polA gene [8 - B. Liu N. Chen CY, Steiner B. New tests for syphilis: rational design of a PCR method for detection of Treponema pallidum in clinical specimens using unique regions of the DNA polymerase I gene. J Clin Microbiol. 2001; 39 (5): 1941-1946], primers 5'-CGTGTGGTATCAACTATGG-3 'and 5'-TCAACCGTGTACTCAGTGC-3' for the bacterium-specific penicillin-binding protein tp47 [9 - Burstain, J. M., Grimprel, E., Luke , SA, Norgard, MV & Radolf, J.D. Sensitive detection of Treponema pallidum by using the polymerase chain reaction. J. Clin. Microbiol. 1991; 29 (1); 62-69], as well as primers for the gene sequences tmpA, tmpC, etc.

However, providing close to 100% specificity for the detection of pathogens of bacterial infections, the use of the above primers does not lead to sufficient sensitivity of the diagnostic test. In particular, when M. leprae is detected, the use of primers for sodA, 16S rRNA, and Ag85B genes demonstrates a detection sensitivity of 59.7–66.1% [10 - Martinez A.N., Ribeiro-Alves M., Sarno E.N., Moraes M.O. Evaluation of qPCR-based assays for leprosy diagnosis directly in clinical specimens. Plos negl. Trop. Dis. 2011; 5 (10): e1354]. When T. pallidum is detected, similar sensitivity indicators are in the range of 55.0-95.2%, depending on the clinical material used for the study [11 - Gayet-Ageron A., Lautenschlager S., Ninet B., Perneger TV, Combescure C. Sensitivity, specificity and likelihood ratios of PCR in the diagnosis of syphilis: a systematic review and meta-analysis. Sex Transm Infect. 2013; 89: 251-256].

In the context of the claimed invention, it is fundamentally important that each bacterial cell “presents” only two fragments of its genome (one for direct and one for reverse primers) for interaction with primers, which puts the PCR efficiency clearly dependent on the quantitative presence of detected bacteria in the analyzed sample. In this regard, as the main reason preventing the obtaining of a positive result from the use of the above methods, a relatively low bacterial contamination of the test material and the resulting insufficient number of sections of the genomic DNA of the detected bacteria complementary to the primers introduced into the sample appear to be. In addition, despite the fact that the theoretically claimed sensitivity of PCR analysis is 10-100 cells in a sample, the real detectable amounts (in the presence of subject residues, anticoagulants and other chemical compounds that reduce the amplification efficiency) are 1-2 orders of magnitude higher.

In this regard, to increase the sensitivity of detection of infectious agents, in addition to the PCR method (DNA amplification), RNA amplification methods have been proposed, which include the NASBA transcriptional amplification method (Nucleic Acid Sequence-Based Amplificaition) [12 - US 5130238 A, Enhanced nucleic acid amplification process ]. Since the “targets” for primer binding using NASBA are ribosomal bacterial RNAs present in each cell in the amount of several hundred to tens of thousands, this method can significantly increase the sensitivity of detection and identify pathogens of bacterial infections in cases where their number is very small and not enough to detect by PCR. However, a significant drawback of this method is the low stability of RNA (unlike DNA), which determines special additional and rather stringent requirements for the procedure for collection, storage and delivery of the test material.

Other methods and approaches to increasing the sensitivity of the polymerase chain reaction when detecting DNA of pathogenic bacteria with a low quantitative content in the test material are not known from the prior art.

The main task to be solved by the claimed method is aimed at increasing the sensitivity of the polymerase chain reaction when detecting DNA of pathogenic bacteria (including Mycobacterium leprae, MTBC and Treponema pallidum), which are typically characterized by a low quantitative content in the studied pathological clinical material.

The essence of the proposed method, formulated at the level of functional generalization and underlying it, is the following:

- the sensitivity of the polymerase chain reaction when detecting the DNA of pathogenic bacteria can be increased by using primers and DNA probes for specific repeating elements of their genome present in the bacterial chromosome in the amount of two or more (up to several tens) copies.

Thus, an original technical solution is the use of multi-copy regions of bacterial genomes as “targets” for binding primers, while observing the specificity requirements of their nucleotide sequences for a particular detectable bacterial species.

Accordingly, when implementing the proposed method, the characteristics of actions, the order of their implementation and the conditions for implementation are presented as follows.

At the preliminary stage, the genomes of the detected bacteria are analyzed in order to search for species-specific repeating (multi-copy) nucleotide sequences in them. This result can be achieved in the following ways: (a) genome-wide sequencing de novo; (b) re-sequencing; (c) bioinformation analysis of genome-wide libraries previously located on accessible electronic resources. Moreover, fragments of the genome, which are subsequently used for amplification, are selected from highly conserved regions, and the same sequences of closely related species should preferably have the most significant differences in the DNA sequence.

After identifying the sequences, the forward and reverse primers are constructed in accordance with the following main criteria: (a) specificity with special emphasis on the nucleotide sequence of the 3'-ends from which the completion of the complementary chain using DNA polymerase begins; (b) the exclusion of the possibility of the formation of primers dimers or loops, leading to erroneous annealing of the primers themselves or with each other. The construction of the DNA probe (TaqMan samples) is carried out in accordance with the following main criteria: (a) specificity of the amplified DNA region; (b) an increased proportion of G + C, providing a melting point 10 ° C higher than that of the primers used, (c) the absence of autofluorescence of the quencher; (d) overlap of the spectrum of the quencher with the spectrum of the fluorescent dye.

In particular, within the framework of the present invention, the use of the following primers and DNA probes is proposed:

- when detecting Mycobacterium leprae DNA, direct 5'-GCAGCAGTATCGTGTTAGTGAA-3 'and reverse 5'-CGCTAGAAGGTTGCCGTAT-3' primers are used, as well as a 5'-CGCCGACGGCCGGGATCATCGA-3 gene-specific DNA probe (repeating gene specific for 29 ' bacterial chromosome, absence of other species of the genus Mycobacterium in the genome);

- when detecting MTBC DNA, direct 5'-AGACGTTATCCASSATAS-3 'and reverse 5'-AGTGCATTGTCATAGGAG-3' primers, as well as a 5'-TCTCAGTACACATCGATCCGGT-3 'DNA probe to a specific repeating element of the MTBC genome from 5 to 20 TB are used. in the bacterial chromosome, the absence in the genome of other species of the genus Mycobacterium);

- when detecting Treponema pallidum DNA, direct 5'-CACTCCTGTGGGGAGTAGGA-3 'and reverse 5'-GAGCTCCCCGTTGCCA-3' primers, as well as a 5'-CGATTACCTGCATCGGCAGGGTC-3 'DNA probe for a specific repeating element of 4 repeating bacterial chromosome, absence of other species of the genus Treponema in the genome).

At the end of the preparatory phase, the synthesis of these primers and DNA probes is carried out. In the synthesis of DNA probes, a fluorophore (for example, carboxyflorescein-FAM) is covalently attached at the 5'-end and a fluorescence quencher (for example, BHQ1) at the 3'-end, the absorption spectrum of which overlaps the FAM fluorescence spectrum. This action makes it possible to carry out the subsequent polymerase chain reaction with the detection of the result in real time (by accumulation of the fluorescent signal).

At the next (main) stage in microtubes or wells of strips (0.2 ml), the analyzed samples are mixed (in a volume of 10 μl) and the components necessary for amplification, namely:

- a mixture of primers (forward and reverse) with a concentration of 10 μM each and a DNA probe with a concentration of 5 μM corresponding to a specific repeating fragment of the detected bacterial genome - 1 μl;

- PCR mixture containing Taq polymerase (5 units / μl), a mixture of nucleotide triphosphates (0.2 mm each), Mg ²⁺ salt (3 mm) - 5 μl;

- deionized water - 9 μl.

Tubes or wells of strips are tightly closed and PCR is performed in a thermal cycler according to the program: initial denaturation - 95 ° С, 5 min; 40 cycles - 95 ° С for 10 sec; 52 ° С for 35 sec; 72 ° C for 1 sec. The essence of the stage is the amplification of specific repeating elements of the bacterial genomes, during which the 5'-3'-exonuclease activity of Taq polymerase leads to cleavage of the FAM fluorophore from the probe, which passes into solution and when excited at 492 nm emits at 520 nm with a quantum yield of ~ 0 ,8.

At the final stage, which overlaps the main stage in time, dynamic registration of fluorescence (at each amplification cycle) is carried out, expressed in relative fluorescence units (OEF) and presented as graphical dependences of the fluorescence signal accumulation on the number of amplification cycles. The final analysis of the PCR result consists in achieving (positive result) or not achieving (negative result) Ct (threshold cycle) - the intersection of the fluorescence signal accumulation curves with the threshold line defined by the instrument software or determined independently by the researcher.

The ability to obtain the desired technical result when using these primers is confirmed by the following set of cause-and-effect relationships.

1. Evaluation of the PCR result of reference DNA samples of M. leprae, MTVS in comparison with DNA samples of closely related microorganisms (non-pathogenic representatives of the genus Mycobacterium), as well as reference samples of T. pallidum DNA in comparison with samples of the genetic material of other microorganisms - sexually transmitted infections potentially present in the studied clinical material together with T. pallidum, allowed to ascertain the high specificity of the proposed primers to the repeating elements of the genome of the detected ba Theurillat. From those shown in FIG. The 1-3 graphical dependences of the accumulation of the fluorescent signal on the number of amplification cycles show that the intersection with the threshold line was achieved for M. leprae, M. tuberculosis and T. pallidum, while in the comparison samples a similar result was not recorded in any case.

In FIG. Figure 1 shows the results of the accumulation of a fluorescent signal for Mycobacterium leprae DNA and the absence of a fluorescent signal for DNA of non-pathogenic representatives of the genus Mycobacterium.

In FIG. Figure 2 presents the results of the accumulation of a fluorescent signal for Mycobacterium tuberculosis and the absence of a fluorescent signal for DNA of non-pathogenic representatives of the genus Mycobacterium.

In FIG. Figure 3 presents the results of the accumulation of a fluorescent signal for Treponema pallidum and the absence of a fluorescent signal for DNA of STI pathogens.

The main reason leading to obtaining such a positive result from the use of the invention is the complementarity of the proposed primers to specific repeating nucleotide sequences of the M. leprae, MTBC and T. pallidum genome in the absence of their pairing with the nucleotide sequences of DNA of other microorganisms.

2. Comparison of the PCR result of reference DNA samples of M. leprae, MTBC and Tpallidum using primers for repeating elements of the genome of detected bacteria, as well as known primers for species-specific genes of these microorganisms present in their genome in the number of single copies, allowed to ascertain the high sensitivity of the proposed method , which consists in reducing the number of amplification cycles required to cross the threshold line.

In FIG. Figure 4 shows that, when testing reference M. leprae DNA samples, the intersection with the threshold line using the direct 5'-GCAGCAGTATCGTGTTAGTGAA-3 ', the reverse 5'-CGCTAGAAGGTTGCCGTAT-3' primers and the 5'-CGCCGACGGGGGGGGGG probe DNA - ML-FRP) was achieved on the 20th amplification cycle, while the use of primers for the species-specific genes MntH, rpoB and fbp allowed to achieve a similar result only on the 36th amplification cycle.

In FIG. Figure 5 shows that when testing a reference sample of M. tuberculosis DNA, the use of direct 5'-AGACGTTATCCACCATAC-3 ', reverse 5'-AGTGCATTGTCATAGGAG-3' primers and 5'-TCCCAGTACACATCGATCCGGT-3 'DNA probe (designation - MTBC-FRP) to the intersection of the threshold line on the 27th amplification cycle, while the use of primers for the rpoB, katG and recX genes allowed us to achieve a similar result on the 33rd amplification cycle.

In FIG. 6 shows that when testing a reference sample of T. pallidum DNA using direct 5-CACTCCTGTGGGGAGTAGGA-3 ', reverse 5'-GAGCTCCCCGTTGCCA-3' primers and 5'-CGATTACCTGCATCGGCAGGGTC-3 'DNA probe (indicated by TP crossing the threshold line on the 29th amplification cycle, the use of primers for the polA gene led to a similar result on the 30th cycle, and the primers for the tp47 gene did not even reach the threshold line to the 40th cycle.

The main reason leading to obtaining such a positive result from the use of the invention is the multiple copying (from 4 to 29 per genome) of specific nucleotide sequences of detected bacteria, providing an increased number of sites for complementary binding of primers during amplification.

The invention is illustrated, but not limited to the following examples.

Example 1

Patient R. was treated in a specialized medical institution of the Russian Federation. The clinical diagnosis according to the extract from the medical history: "Leprosy, lepromatous type (LLs), active stage." During the treatment period, a clear regression of the clinical manifestations of the disease has not been achieved.

For the control study, the following clinical biomaterial was used:

- skin biopsy (thigh, buttock);

- Scarificate (earlobe, superciliary arch, forearm, back, buttock, patella);

- scraping from the nasal mucosa.

In order to confirm the presence of M. leprae in the analyzed samples, a PCR study was carried out using a mixture of primers and the ML_FRP DNA probe created in accordance with the claimed method. The results of the PNR study are shown in FIG. 7, which shows the curves of fluorescence signal accumulation for DNA samples of clinical material (biopsy, scarificates, scrapings from the nasal mucosa). The obtained data were compared with the results of the study of the same clinical material using the commercial Genesig Standard Kit (Primerdesign Ltd, UK), based on the use of a mixture of primers and a DNA probe for the rpoB gene region. The results of a PCR assay using the Genesig Standard Kit are shown in FIG. 8, where the fluorescence signal accumulation curves are shown for DNA samples of clinical material (biopsy, scarificates, scrapings from the nasal mucosa).

As a result of using a mixture of primers and the ML_FRP DNA probe, a positive PCR result was obtained in the study of all clinical samples. When conducting a study using a commercial kit based on the amplification of the rpoB gene, PCR results with biopsy samples and scarificates were also positive, however, the result of the analysis of scrapings from the nasal mucosa was negative. Using a mixture of primers and the ML_FRP DNA probe, the resulting Ct range varied from 20 to 27 (for biopsy specimens and scarificates) and Ct 30 (for a scraping sample from the nasal mucosa). The use of a commercial kit based on the amplification of the rpoB gene made it possible to obtain Ct from only 30 to 38 (for biopsy specimens and scarificates) without reaching the threshold line for a sample of scraping from the nasal mucosa.

Thus, the PCR in accordance with the claimed method (using primers and a DNA probe to a repeating element of the M. leprae genome) in comparison with the known method based on the use of primers and a DNA probe to the rpoB gene represented in the M. leprae genome in a single copy, it increased the sensitivity of the study.

Example 2

When analyzing control samples provided by the ANSNP “Center for External Quality Control of Clinical Laboratory Research” (Russia), the use of a mixture of primers and a DNA probe MTBCFRP created on the basis of the proposed method allowed to obtain a positive PCR result for 10 out of 12 test samples (DNA of mycobacteria of the MTVS group was detected ); the reaction with 2 samples turned out to be negative (lack of DNA from mycobacteria of the MTVS group). The results are shown in FIG. 9, which shows the accumulation of a fluorescent signal for 10 Mycobacterium tuberculosis DNA samples and the absence of fluorescence signal accumulation for Mycobacterium avium DNA.

Comparison of the obtained data with the test results of the same control samples using the TB-BIOCHIP DNA chip developed by the FSBI Institute of Molecular Biology V.A. Engelhardt "RAS, allowed us to confirm the coincidence of the presence of Mycobacterium tuberculosis DNA in 10 of 12 test samples, while Mycobacterium avium DNA was detected in 2 PCR-negative samples.

Thus, PCR in accordance with the claimed method (using primers and a DNA probe to a repeating element of the MTVS genome) made it possible to achieve a specific and sensitive detection result.

Example 3

Patient K. was examined in a specialized medical institution of the Russian Federation. The clinical diagnosis according to the extract from the medical history: “Primary syphilis of the genital organs” (based on clinical examination), however, due to the rather late period of detection of this disease, the study of the chancroid was negative.

For the study, the following clinical biomaterial was used:

- blood plasma.

In order to confirm the diagnosis, a study was conducted using a mixture of primers and TP_FRP DNA probe created on the basis of the proposed method. The data obtained are compared with the results of the study of the same clinical biomaterial using the AmpliSens Treponema pallidum-FL commercial kit, based on the use of a mixture of primers and a DNA probe for the tp47 gene region represented in a single copy of the T. pallidum genome.

When conducting PCR analysis, a positive result was obtained using both the claimed and the known set of primers and a DNA probe, which made it possible to confirm the clinical diagnosis of patient K. However, as shown in Fig. 10, the obtained Ct 30 values in the case of using a mixture of primers and The TP_FRP DNA probe in comparison with Ct 32 when using a commercial reagent kit based on the amplification of the tp47 gene, allows us to conclude that the PCR analysis is more sensitive in accordance with the claimed method.

Claims (4)

1. A method for detecting DNA of Mycobacterium leprae, Mycobacterium tuberculosis complex and Treponema pallidum using a real-time polymerase chain reaction, characterized in that, in order to increase the sensitivity of the method, oligonucleotides complementary to specific repeating elements of the detected bacteria genome present are used as primers and probes in the bacterial chromosome in the amount of four or more copies. 2. The method according to claim 1, characterized in that when detecting Mycobacterium leprae DNA, direct 5'-GCAGCAGTATCGTGTTAGTGAA-3 ', reverse 5'-CGCTAGAAGGTTGCCGTAT-3' primers and a 5'-CGCCGACGGCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG test probe are used. genome of M. leprae, having 29 repeats in the bacterial chromosome. 3. The method according to p. 1, characterized in that when detecting DNA of Mycobacterium tuberculosis complex, direct 5'-AGACGTTATCCACCATAC-3 ', reverse 5'-AGTGCATTGTCATAGGAG-3' primers and 5'-TCTCAGTACACATCGATCCGGT-3 'DNA probe are used an element of the genome of Mycobacterium tuberculosis complex, having from 5 to 20 repeats in the bacterial chromosome. 4. The method according to p. 1, characterized in that when detecting Treponema pallidum DNA, direct 5'-CACTCCTGTGGGGAGTAGGA-3 ', inverse 5'-GAGCTCCCCGTTGCCA-3' primers and a 5'-CGATTACCTGCATCGGCAGGTC-specific element probe are used. genome of T. pallidum, having 4 repeats in the bacterial chromosome.

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