RU2746100C1 - Method of multiplex detection of toxin genes - Google Patents

Abstract

FIELD: medicine; microbiology.SUBSTANCE: method of multiplex detection of toxin genes is proposed. At the first stage, DNA-libraries synthesis is carried out. At the second stage, the targeted enrichment of DNA-libraries molecules carrying sections of the analyzed toxin genes is carried out using method of liquid hybridization of DNA-probes. Quantification of the amplified DNA-libraries is carried out for the following NGS sequencing using fluorimetric method.EFFECT: invention provides increase in relative representation of target sections of the toxin genes in the final DNA mix analyzed with high-performance sequencing methods in compare with incoming DNA.1 cl, 1 tbl

Description

The technical field to which the invention relates

The invention relates to the field of research in medicine, microbiology. This invention can be used to identify genes of toxins for the protection of humans or animals, in order to search for bacteria included in the list determined by sanitary rules in relation to 3-4 groups of pathogenicity, and to clarify the profile of their danger by the content of genes of toxins, genes of antibiotic resistance and genetic engineering vectors for use as part of the network of the National Integration Center for Monitoring Biological Threats.

State of the art

A method of species and strain identification of bifidobacteria of the phylotype Bifidobacterium longum is known from the prior art, characterized in that it is based on a combination and polymorphism of the toxin-antitoxin genes of the RelBE superfamily and is characterized in that for identification, amplification is carried out with genomic DNA using a set of species- and strain-specific oligon PCR products are analyzed in an agarose gel, and the size of the resulting fragment is determined using a DNA marker, species identification is carried out using specific oligonucleotides, and the studied strains are classified as B. longum in the case of a target fragment of 275 bp; strain identification is carried out using oligonucleotides, the investigated strain is recognized as identical to the strain B. longum subsp.infantis ATCC 15697 in the case of the production of fragments when using oligonucleotides ReB1N-ReB1C, ReB2N-ReB2C and ReB3.1N-ReB3.2C; the investigated strain is recognized as identical to the B. longum subsp.infantis ATCC 55813 strain in the case of the accumulation of fragments when using the oligonucleotides ReB3.1N-ReB3.1C and ReB9.1N-ReB9.1C; the investigated strain is recognized as identical to the B. longum subsp.infantis CCUG 52486 strain in the case of the accumulation of fragments when using the oligonucleotides ReB3.1N-ReB3.1C, ReB3.4N-ReB3.4C and ReB9.1N-ReB9.2C; the investigated strain is recognized as identical to the B. longum DJO10A strain in the case of the accumulation of fragments when using oligonucleotides ReB3.1N-ReB3.1C; the investigated strain is recognized as identical to the Bifidobacterium sp. (B. longum) 12_1_47BFAA strain in the case of the accumulation of fragments when using the oligonucleotides ReB3.1N-ReB3.1C and ReB4N-ReB4C; the investigated strain is recognized as identical to the B. longum subsp.longum F8 strain in the case of the accumulation of fragments when using the oligonucleotides ReB2N-ReB2C and ReB3.3N-ReBC; the investigated strain is recognized as identical to the B. longum NCC2705 strain in the case of the accumulation of fragments when using oligonucleotides ReB3.1N-ReB3.2C; the investigated strain is recognized as identical to the B. longum subsp.longum JDM301 strain in the case of the accumulation of fragments when using oligonucleotides ReB8N-ReB8C, ReB2N-ReB2C and ReB3.1N-ReB3.1C; the investigated strain is recognized as identical to the B. longum subsp.longum BBMN68 strain in the case of the accumulation of fragments when using the oligonucleotides ReB3.1N-ReB3.1C and ReB3.4N-ReB3.4C; the investigated strain is recognized as identical to the B. longum subsp.infantis 157F strain in the case of the accumulation of fragments only when using the oligonucleotides ReB3.1N-ReB3.1C, ReB4N-ReB4C and ReB5N-ReB5C; the investigated strain is recognized as identical to the B. longum subsp.longum JCM 1217 strain in the case of the accumulation of fragments when using the oligonucleotides ReB3.1N-ReB3.1C and ReB3.5N-ReB3.4C; the investigated strain is recognized as identical to the B. longum subsp.longum strain KASS 91563 in the case of the accumulation of fragments when using oligonucleotides ReB10N-ReB10C and ReB3.1N-ReB3.4C (RU 2527069 C2, 27.08.2014).

It is known in the art to create and screen gene libraries http://www.bio.bsu.by/molbiol/files/lections/Razdel4.pdf. The specified source of information discloses the receipt of DNA libraries: a genomic library is a collection of all nucleotide sequences of the genomic DNA of an organism, cloned as fragments in vectors. With subsequent screening using the method of colony hybridization, immunodetection of gene products. In addition, obtaining linked libraries is disclosed - the chromosome walk method. And also disclosed and sequencing - the definition of the primary structure (sequence of nucleotides) of the DNA molecule. This information source was chosen as a prototype.

The disadvantages of the previously known methods are their low productivity, the complexity of the analysis and selectivity. For example, when using these oligonucleotides, more than 10 specific amplifications may be required to detect one strain of Bifidobacterium longum.

The era of the discovery of new pathogenic microorganisms, not only has not ended, but on the contrary, thanks to new opportunities, it has acquired the opportunity to study evolutionary and epidemically significant mechanisms of variability, which allow pathogens to threaten humanity with a biological catastrophe more sophisticatedly and with greater dynamics. Each new infectious disease in the 21st century, like never before, can be a serious challenge for modern medicine and jeopardize the safety of the population of certain territories and sovereign states. Environmental change, deforestation of tropical forests, ice melting in Antarctica, an increase in population density and the widespread use of antibiotics - all this is not a complete list of relevant factors provoking the emergence of new pathogens and giving pathogens new, epidemically significant properties. Influenza epidemics caused by new strains of the virus; respiratory syndromes caused by new coronaviruses; outbreaks of infections caused by hemolytic Escherichia coli; the emergence of antibiotic-resistant superbugs are examples of the dangers that may await humankind. Developing ways to quickly and accurately identify them is a priority to address emerging pathogens. Classical methods widely used for typing microorganisms in laboratories, such as PCR, ELISA and MAPDI, have limitations in the volume and quality of data that can be obtained on the properties of infectious agents contained in the sample of interest. Thus, there is an urgent need to develop methods that allow the detection and identification of new pathogens using next generation sequencing (NGS) methods.

Implementation of the invention

The claimed method includes the following techniques:

1) at the first stage, DNA libraries are created from the isolated genomic DNA of bacteria for subsequent sequencing on high-throughput sequencing platforms. This stage includes the following stages:

• enzymatic fragmentation of DNA;

• end repair to obtain double-stranded 5'-phosphorylated DNA with protruding 3'-adenosine;

• ligation of specific adapters using fast TA-ligation technology;

• amplification of the obtained DNA libraries from external universal adapter sequences;

• selection of DNA libraries of target length using silicate paramagnetic particles.

The use of various specific ligation adapters allows multiplexing of images for co-sequencing within a single platform run. Variations in the selection method using magnetic particles allows varying the length of the obtained DNA libraries for analysis using high-throughput sequencing on various NGS and TGS platforms (including Illumina, Ion Torrent, BGI, Oxford Nanopore, etc.).

2) the second stage includes the targeted enrichment (selection) of DNA library molecules carrying regions of the analyzed toxin genes using the DNA probe liquid hybridization method. Structurally optimized synthetic single-stranded DNA molecules (oligonucleotides) are used as probes, carrying a modification at the 5 'or 3' end that allows them to reversibly bind to specialized paramagnetic silicate particles (for example, in the form of a modification of biotin, an alkyne group, an azide group, and similar groups click chemistry). Oligonucleotide probes in appropriate buffer and temperature conditions form, together with target DNA molecules, stable complementary pairs (duplexes). These target duplexes are subsequently associated with superparamagnetic particles containing groups that are reactive with respect to modifications of synthetic probes. For example, the biotin molecules of the probes form a covalent bond with streptavidin on the surface of the magnetic particles, as a result of which the DNA molecules that formed the DNA probe duplexes are bound to the magnetic particles. Next, the unbound non-target DNA molecules are washed. Whole DNA molecules are subsequently released from the surface of the magnetic particles and can be analyzed by NGS sequencing methods.

This stage includes the following stages:

• liquid hybridization of synthesized DNA libraries with probes;

• capture of hybrid duplexes using superparamagnetic particles;

• washing of unbound non-target DNA molecules;

• release of bound DNA libraries from the surface of magnetic particles;

• PCR amplification of enriched DNA libraries;

• quantification of amplified DNA libraries for subsequent NGS sequencing.

The objective of the present invention is the highly efficient isolation of target groups of toxin genes from complex mixtures of heterogeneous input DNA (mixtures of genomic DNA of various composition and species origin) with subsequent targeted high-throughput sequencing on various NGS and TGS platforms (including Illumina, Ion Torrent, BGI, Oxford Nanopore and others) to identify fragments of genes of toxins present in the initial mixture.

The technical result consists in a significant increase in the relative representation of target fragments of toxin genes in the final mixture of DNA, analyzed by high-throughput sequencing methods, in comparison with the incoming DNA (the so-called enrichment of target fragments). For example, as a result of applying the method to a mixture of genomic DNA of toxigenic bacterial strains, the representation of target fragments increases from 0.001% in the initial mixture to 50% or more in the final analyzed sample.

Disclosure of invention

The indicated technical result is realized through the following methods using the example of sample preparation for the Illumina platform.

The method for multiplex detection of toxin genes is implemented as follows: at the first stage, DNA libraries are prepared, while this stage includes the following techniques:

• perform enzymatic fragmentation of DNA using non-specific exonucleases;

• carry out the restoration of the ends in order to obtain double-stranded 5'-phosphorylated DNA with protruding 3'-adenosine;

• carry out ligation of specific Y-shaped adapters using fast TA-ligation technology using T4-DNA l and gases, or similar;

• carry out amplification of the obtained DNA libraries using primers complementary to the external sequences of specific Y-shaped adapters;

• selection of DNA libraries of target length using silicate paramagnetic particles;

at the second stage, the targeted enrichment of DNA library molecules carrying portions of the analyzed toxin genes is performed using the DNA probe liquid hybridization method, while this second stage contains the following techniques:

• carry out liquid hybridization of the synthesized DNA libraries with probes, while mixing DNA libraries with specific probes to enrich fragments of target toxin genes using commercially available buffers for hybridization (for example, HybBuf, Roche). The mixture for hybridization is incubated in a solid-state thermostat or amplifier at a temperature of 35 ° C to 60 ° C for 10-50 hours;

• carry out the capture of hybrid duplexes using superparamagnetic particles that bind the duplexes of the DNA probe, while mixing the solution after hybridization with superparamagnetic particles;

• perform washing of unbound non-target DNA molecules, while performing a series of washes of superparamagnetic particles carrying bound DNA probe duplexes, using solutions to remove unbound non-target DNA molecules;

• carry out the release of bound DNA libraries from the surface of magnetic particles, for which enriched target DNA fractions are separated from superparamagnetic particles by heating the mixture at 95 ° C for 10-15 minutes;

• then carry out PCR amplification of enriched DNA libraries from external universal primers complementary to external sequences of specific Y-shaped adapters;

• after that, the amplified DNA libraries are quantified for subsequent NGS sequencing by fluorimetric and / or spectrophotometric methods.

Detailed disclosure of the implementation of the invention

At the first stage, DNA libraries are created. For this, the DNA isolated from the test sample is fragmented, the fragmented DNA is purified, the ends are reconstructed and the adapters are ligated, amplified from flanking primers, followed by purification on magnetic particles. Subsequently, the prepared libraries are normalized and combined for subsequent enrichment.

Moreover, this stage includes the following stages:

• enzymatic fragmentation of DNA using nonspecific exonucleases;

• restoration of the ends in order to obtain double-stranded 5'-phosphorylated DNA with protruding 3'-adenosine;

• ligation of specific adapters (for the selected sequencing platform) using rapid TA ligation technology using T4-DNA ligase, or similar;

• amplification of the obtained DNA libraries from external universal adapter sequences;

• selection of DNA libraries of target length using silicate paramagnetic particles.

The use of different ligation adapters allows images to be multiplexed for co-enrichment and sequencing within a single platform run. At the stage after ligation of adapters (before PCR), DNA libraries can be selected by length (two-sided size-select) using magnetic particles (AMPure XP, Beckman Coulter, or similar) to obtain libraries with an insert of the required length. For example, it is possible to select a fraction of libraries with an average insert length of 450-500 bp, which corresponds to a total library length of 570-620 bp. taking into account the length of the adapter sequences. This length of DNA libraries is optimal for subsequent sequencing on the Illumina MiSeq or HiSeq platform with a read length of 2 × 250 bp. Variations in the selection method using magnetic particles allows varying the length of the obtained DNA libraries for analysis using high-throughput sequencing on various NGS and TGS platforms (including Illumina, Ion Torrent, BGI, Oxford Nanopore, etc.).

At the second stage, a targeted enrichment (selection) of DNA library molecules carrying portions of the analyzed toxin genes is performed using the “DNA probe” liquid hybridization method. Structurally optimized synthetic single-stranded DNA molecules (primers) carrying a modification in the form of a biotin molecule at the 5 'or 3' ends are used as probes. DNA probes in appropriate buffer and temperature conditions form, together with target DNA molecules, stable complementary pairs (duplexes). These target duplexes subsequently bind to streptavidin-coated superparamagnetic particles. The biotin molecules of the probes form a covalent bond with streptavidin on the surface of the magnetic particles, as a result of which the DNA molecules that formed the DNA-probe duplexes are bound to the magnetic particles. Next, the unbound non-target DNA molecules are washed. Whole DNA molecules are subsequently released from the surface of the magnetic particles and can be analyzed by NGS sequencing methods. The probe panel was synthesized in the form of 5'-biotinylated oligonucleotides (see Table 1) by solid-phase synthesis on an ABI 3900 automatic synthesizer. This panel was used to test the enrichment technique.

This second stage includes the following stages:

• liquid hybridization of synthesized DNA libraries with probes. Within the framework of this stage, DNA libraries are mixed with specific probes for hybridization in the presence of the necessary buffer conditions. The hybridization mixture is incubated in a solid-state thermostat or thermostat at a selected temperature for 10-50 hours.

• capture of hybrid duplexes using specific superparamagnetic particles that bind DNA probe duplexes. Within the framework of this stage, the solution is mixed with specific superparamagnetic particles after hybridization.

• washing of unbound non-target DNA molecules. As part of this stage, a series of washes of superparamagnetic particles carrying the bound DNA probe duplexes takes place, using solutions to remove unbound non-target DNA molecules.

• release of bound DNA libraries from the surface of magnetic particles. Within the framework of this stage, the enriched target DNA fraction is separated from the superparamagnetic particles. For example, by heating the mixture at 95 ° C for 10-15 minutes.

• PCR amplification of enriched DNA libraries from external universal adapter sequences;

• quantification of amplified DNA libraries for subsequent NGS sequencing by fluorometric and / or spectrophotometric methods.

The finished DNA libraries were hybridized using the developed probe panel and SeqCap EZ Hybridization and Wash Kit (Roche). The samples were subjected to the following procedure: hybridization of DNA libraries with probes at 40 ° C for 24-48 hours, washing of unbound DNA, post-hybridization amplification from external flanking primers. The SeqCap EZ Hybridization and Wash Kit includes oligonucleotide blocking buffers, primers and polymerase for all hybridization and PCR steps.

Industrial applicability

The claimed method can be carried out by a specialist in practice and, when implemented, ensures the implementation of the declared purpose. The possibility of implementation in practice follows from the fact that for each feature included in the claims based on the description, a material equivalent is known, which allows us to conclude that the criterion "industrial applicability" for the invention and the criterion "completeness of disclosure" for the invention are met.

METHOD FOR MULTIPLEX DETECTION OF TOXIN GENES

LIST OF SEQUENCES

Claims (13)

The method for multiplex detection of toxin genes is implemented as follows: at the first stage, the synthesis of DNA libraries, while this stage includes the following techniques: - perform enzymatic fragmentation of DNA using exonucleases; - carry out the restoration of the ends in order to obtain double-stranded 5'-phosphorylated DNA with protruding 3'-adenosine; - carry out ligation of Y-shaped adapters using the technology of rapid TA-ligation using T4-DNA ligase; - carry out the amplification of the obtained DNA libraries using primers complementary to the external sequences of the Y-shaped adapters; - perform selection of DNA libraries of target length using silicate paramagnetic particles; at the second stage, the targeted enrichment of DNA library molecules carrying portions of the analyzed toxin genes is performed using the DNA probe liquid hybridization method, while the specified second stage includes the following techniques: - carry out liquid hybridization of the synthesized DNA libraries with probes, while mixing DNA libraries with probes for hybridization, while the mixture for hybridization is incubated in an amplifier at a temperature of 35 ° C to 60 ° C for 10-50 hours; - carry out the capture of hybrid duplexes using superparamagnetic particles that bind duplexes of the DNA probe, while mixing the solution after hybridization with superparamagnetic particles; - perform washing of unbound non-target DNA molecules, while performing a series of washes of superparamagnetic particles carrying bound DNA probe duplexes, using solutions to remove unbound non-target DNA molecules; - carry out the release of bound DNA libraries from the surface of magnetic particles, for which enriched target DNA fractions are separated from superparamagnetic particles by heating the mixture at 95 ° C for 10-15 minutes; - then carry out PCR amplification of the enriched DNA libraries using primers complementary to the external sequences of the Y-shaped adapters; - perform quantification of amplified DNA libraries for subsequent NGS sequencing by fluorometric method.