RU2397251C1 - Set for detecting legionellosis agent in biological material and environmental medium - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology. The invention seeks to create a set for detecting a legionellosis agent in biological material and environmental medium, with possibility of its use in a polymerase chain reaction with electrophoretic and hybridisation-fluorescent reading of results. The set for detecting a L. pneumophila agent contains species-specific primers Lp1 - 5'-GCTTGGTGCTGTGAAAGGTA-3', Lp2 - 5'-CCTCCAATAAAGGAGATGCAA-3' and probe Lp-3 - 5'-ROX-TCACCGTACGCACATCCGCA-BHQ2-3'. The DNA target for specific amplification of L. pneumophila is a DNA fragment of the fisZ gene lying in the range from 397 to 758 of the nucleotide complete locus sequence.

EFFECT: invention enables fast and highly specific detection of L pneumophila of 1-14 serogroups in clinical samples and environmental medium samples.

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Description

The invention relates to the field of biotechnology and can be used to identify the causative agent of legionellosis in biological material and environmental objects in practical health care and the Rospotrebnadzor service.

Legionellosis is one of the dangerous infectious diseases occurring in the form of severe pneumonia, in which up to 15% of cases are fatal, or in the form of respiratory diseases.

Most cases of legionellosis are associated with the species Legionella pneumophila, however, the most dangerous are Legionella pneumophila I serogroups (1). Other legionella species, such as L. bozemanii, L. longbeachae, L. micdadei, etc., cause diseases mainly in individuals with reduced immune status (2).

Legionella multiply inside macrophages and are localized in the “indigestible” phagolysosome, therefore antibacterial drugs are used for treatment that can penetrate cell membranes and accumulate inside vacuoles, which distinguishes treatment for pneumonia caused by other pathogens (3).

In this regard, early diagnosis in clinical samples of the causative agent of legionellosis allows you to adjust the patient’s treatment and avoid serious consequences, and timely and quick detection of it in environmental objects helps to prevent epidemic outbreaks.

The efficacy of patient management and epidemiological surveillance of Legionella spp. is largely determined by the quality of laboratory diagnostics. For the diagnosis of legionellosis, bacteriological, immunological, molecular genetic research methods are used.

The bacteriological method for diagnosing legionellosis is the “gold standard”, but its sensitivity is on average no more than 60%, despite the use of high-quality nutrient media (4). In addition to low sensitivity, the disadvantage of the cultural method is its duration. The maximum number of visible colonies grows by 5-8 days.

Immunological research methods have a fairly high sensitivity, for example, using commercial ELISA test systems, it is possible to detect the antigen Legionella pneumophila I of the serogroup, the sensitivity of the method is up to 97%.

Immunochromatographic analysis (IHA) allows the detection of soluble legionella antigen in the urine, and the sensitivity reaches 95% (5, 6, 7). The disadvantage of this method is that it cannot be applied to other clinical material (sputum, blood, lavage, etc.) and samples from environmental objects.

The main disadvantage of commercial ELISA and IHA test systems is that they are not designed to diagnose infections caused by Legionella pneumophila 2-16 serogroups and Legionella spp. (8).

At present, molecular genetic research methods based on the detection of the pathogen DNA in the test material are considered the most promising. Using PCR, it is possible to study biological material - sputum, bronchoalveolar lavage, pleural fluid, oropharyngeal swabs, blood, micro and macroautopaths, as well as samples from environmental objects - water, washes, biofilm samples, as well as pure cultures of the pathogen.

An important step in the development of PCR test systems is the selection of targets for the detection of the pathogen.

Known for use as targets for the detection of Legionella spp. and L. pneumophila fragments of the mip, 16S rDNA, 5S rDNA, 23S-5S, dotA genes for PCR, taking into account the results by electrophoresis (PCR-EF) (9, 10). The described targets do not provide a sufficient level of reliability for the clinical diagnosis of legionellosis during PCR-EF (11).

A careful study of the nucleotide sequences of the Legionella pneumophila genome revealed that the variability of the 16S rDNA gene fragment of 1434 bp in strains of Legionella pneumophila was 2.0-6.0% (an average of 31 polymorphic nucleotide) between L. pneumophila and other legionella species. In the study of 23S-5S genes, the variability among L. pneumophila strains was 7.4%, and between L. pneumophila and other species - 14.0-20.0%. The 23S (143 bp) and 5S (91 bp) genes were highly homologous, while the 23S-5S intergenic region (102 bp) was characterized by a high content of polymorphic nucleotides. Based on the data obtained, it is clear that the use of these targets in PCR is advisable only when using a system of primers and probes, taking into account the results in the "real" time (PCR-RV).

Known kit for the detection of Legionella spp., Containing primers for amplification, for internal control and a probe that allows the identification of the pathogen using PCR-RV (12).

The well-known commercial IQ-Check kit (BioRad) for screening legionella in the environment, designed by American researchers (13). However, the described kit is not intended to identify the causative agent of legionellosis in biological material (sputum, bronchoalveolar lavage, pleural fluid, oropharyngeal swabs, blood, micro and macro autopsies). In addition, the high cost of recruitment and the inability to perform PCR with electrophoretic consideration of the results limit its use in practical laboratories, since PCR, taking into account the results in “real time” mode, involves the use of expensive equipment.

A search by patents and scientific and technical sources of information showed a lack of information on kits designed to detect L. pneumophila by PCR with electrophoretic and hybridization-fluorescence results, which ensure the detection of the pathogen in biological material and environmental objects, as well as available for work in laboratories of Russia. Thus, there is a need to create an inexpensive, sensitive and specific kit that would be widely used in practical health care.

The objective of the invention is to provide a kit for the detection of the causative agent of legionellosis in biological material and environmental objects with the possibility of using it in PCR with electrophoretic and hybridization-fluorescence-based results.

The technical result of the claimed invention is a highly specific and rapid detection of L. pneumophila 1-14 serogroups in biological material and environmental objects.

The technical result is achieved by a kit for the detection of the causative agent of legionellosis. The inventive kit contains species-specific primers Lp1 - 5'-GCTTGGTGCTGTGAAAGGTA-3 ', Lp2 - 5'-ССТССААТAAAGGAGATGCAА-3' and a probe Lp-5'-ROX-TCXCACCgts of f 'L. B-amplification of f' L. Bac pneumophila ranging from 397 to 758 nucleotides of the complete sequence of a locus having the nucleotide sequence of SEQ ID NO 1.

When designing a kit for the detection of the causative agent of legionellosis, particular importance was attached to the selection of new DNA targets.

Based on the analysis of the nucleotide sequence of the genome of the causative agent of L. pneumophila, 10 genes involved in the activity of the cell (house keeping) were tested, and a fragment of the ftsZ gene was selected. Comparative data of the nucleotide sequences of 10 genes (life support) of L. pneumophila and other legionella species are presented in Tables 1 and 2. Analysis of the nucleotide sequences of these genes of L. pneumophila and other legionella species is presented on the GenBank NCBI genetic database. Sequencing of a fragment of the ftsZ gene in L. pneumophila strains isolated in the Russian Federation and their comparison with the nucleotide sequence of similar sites in reference strains of L. pneumophila Philadelphia 1, Corby, Paris, Lens (GenBank NCBI: NC _- 002942, CP 00675, NC _- 006368, NC _- 006369) showed that the most promising for the selection of primers in the development of PCR is a fragment of the ftsZ gene encoding the synthesis of tubulin-like protein (from 397 to 758 nucleotides). As a result of a thorough quality control of the determination of the nucleotide sequence, the consensus sequence of the ftsZ gene fragment SEQ ID NO 1 was deduced:

Justification for the selection of primers and probe.

In the next step, a set of oligonucleotide primers specific for the detection of L. pneumophila was constructed. Based on the consensus sequence of the selected fragment of the ftsZ gene using the Primer Premier-V5 program (Premier Bio Soft International) and the BLAST algorithm, oligonucleotide primers were selected to amplify a 185 bp fragment of this locus. The choice of primers was carried out taking into account the possibility of recording the results both in the "real time" mode and using electrophoresis. To use the primers in PCR-RV, a TaqMan (Lp-3) format probe was selected in online mode at www.genscript.com. Selected probe Lp-z - 5'-ROX-TCACCGTACGCACATCCGCA-BHQ2-3 'to the fisZ gene region and primers: Lpl - 5'-GCTTGGTGCTGTGAAAGGTA-3', Lp2 - 5'-CCTCCAATAAAGGAGATGCAA high specificity and high specificity 3 ' causative agent of legionellosis in samples from environmental objects and biological material.

The specificity of the primers was evaluated by multiple sequence comparison using software such as BLAST (Lastaew to evaluate polymorphisms in the compared sequences).

These primers do not form secondary structures (hairpins) and dimers, which contributes to their effective binding to matrix DNA.

To determine the analytical sensitivity of PCR, quantitatively characterized dilutions of recombinant plasmids with a cloned fragment of the ftsZ gene were used, as well as DNA preparations isolated from samples containing L. pneumophila of various serogroups at a concentration of 1 × 10 ³ -1 × 10 ⁵ MK / ml. The results are presented in figures 1 and 2.

To characterize the specificity of PCR, a selection of strains from the target collection of various microorganisms was used. The specificity of PCR with primers based on the nucleotide sequence of the ftsZ gene is presented in table 3.

As a result of the assessment, it was found that the selected primers and probe for the detection of L. pneumophila have high sensitivity - 1 · 10 ³ MK./ml and a specificity of 100%.

The optimal parameters of the amplification reaction with oligonucleotide primers and the primer-probe system were selected:

- concentration of primers - from 5 to 16 pM;

- probe concentration - from 1 to 5 pM;

- concentration of MgCl - from 2 to 3 pM;

- concentration of Tag polymerase - from 1 to 1.5 units;

- the temperature at which the level of fluorescence in the samples is determined is from 54 to 60 ° C;

- the number of cycles during which fluorescence is read, from 30 to 40;

- temperature annealing of primers - from 50-64 ° C;

- the time of each step of the cycle is 30-60 s;

- number of cycles: 35-40;

- pre-denaturation time 1-5 minutes;

- time of the final elongation of 1-7 minutes

The optimal composition of the reaction mixture for PCR-EF was experimentally established. The selected concentration of primers Lp1 and Lp2, the enzyme Tag polymerase at a concentration of 1.0 units and the Lp-z probe is necessary and sufficient to ensure high specificity and efficiency of the reaction and allow to obtain easily detectable amounts of amplicons. The invention is confirmed by examples.

Example 1. Detection of the DNA of the pathogen L. pneumophila in biological material (sputum, bronchoalveolar lavage, pleural fluid, smears from the oropharynx, blood, micro and macro autopsy samples) using PCR with electrophoretic results.

To identify the causative agent of legionellosis, the biological material is decontaminated by adding 300 μl of a solution containing 6 M guanidine thiocyanate to the prepared samples and heated for 15 minutes at a temperature of 64 ° C.

DNA isolation. 30 μl of the sorbent is added to the disinfected samples, then they are incubated for 10 min at room temperature, periodically shaking on a vortex for 10 s, centrifuged at 12000 rpm for 20 s. The supernatant is taken and 300 μl of the solution is added to the precipitate to wash the sorbent from cell debris and other substances that can inhibit the amplification reaction; mix and centrifuge at 12,000 rpm for 20 s. The supernatant is removed, and 1 ml of the second washing solution (10 mM Tris, 50 mM NaCl, 50% ethanol) is added to the precipitate, stirred and incubated at 64 ° C. The supernatant is removed, the precipitate is washed with 400 μl of acetone, centrifuged at 12000 rpm for 20 s. The precipitate is dried for 10 min at a temperature of 64 ° C, add 50 μl of TE buffer and incubated at a temperature of 64 ° C for 10 min, periodically shaking on a vortex, centrifuged at 12,000 rpm for 2 min; supernatant is used in PCR.

For PCR in 0.5 ml microtubes, a general PCR mixture is prepared on the basis that 10 μl of the reaction mixture and 0.2 μl of the Tag polymerase enzyme are needed per sample. The resulting mixture was thoroughly mixed in a microcentrifuge / shaker and 10 μl was added to microtubes containing a mixture of primers and dNTP under a layer of paraffin. Then, 10 μl of DNA isolated from the studied samples is introduced into the tubes. A negative control tube contains 10 μl of TE buffer, and a positive control tube contains 10 μl of L. pneumophila DNA PCO. Microtubes containing control and experimental samples are transferred to a thermal cycler preheated to 95 ° C and amplification is carried out according to the selected program.

Amplification products are detected by electrophoresis in a 1.5-2.5% agarose gel according to the method described in the manuals of L.A. Osterman and T. Maniatis (14.15). The results are presented in table.3.

Example 2. Detection of the pathogen L. pneumophila in environmental objects (water, swabs, biofilm samples).

Identification of the causative agent of legionellosis from environmental objects is carried out analogously to example 1. The results of detection of the causative agent of legionellosis in biological material are presented in table 4, and in samples from environmental objects in table 5.

In parallel with the use of the inventive kit, samples were studied by other methods. The data obtained showed a high percentage of coincidence of the results.

Example 3. Detection of L. pneumophila in samples artificially contaminated by a microorganism of various serogroups.

DNA isolation and PCR was carried out analogously to example 1. The results are presented in table.6.

The described invention is not limited to the objects indicated in the tables, which are intended only for illustration. Any clinical samples and environmental objects are within the scope of the present invention.

Based on the selected primers and probe, test systems “L. pneumophila gene - REF” and “L. pneumophila gene - RGF” were designed to indicate L. pneumophila. Test systems have been tested on the basis of the Stavropol Scientific Research Institute for Scientific and Technical Research and State Research Center of Virology and Biotechnology "Vector" and are at the stage of implementation in production.

Thus, the use of a new target and the corresponding sets of primers and probe for them for the detection of L. pneumophila 1-14 serogroups by PCR with electrophoretic and / or hybridization-fluorescence analysis of the results provides early diagnosis of the pathogen (within 3-4 hours), which contributes to the effectiveness of treatment of patients, improves the quality of epidemiological surveillance of environmental objects. The proposed set of primers and probe is available for any practical healthcare laboratory, as it allows you to perform studies at various levels of equipment and reagents.

Table 3 View amount results strains PCR-REF PCR-RHF L. pneumophila 22 + + 1-14 serogroups L. dumoffi one - - L. longbeachae one - - L. micdadei one - - Aeromonas spp. 3 - - A. faecalis 3 - - B. anthracis one - - B. cereus 2 - - B. subtilis one - - Comamonas spp. 3 - - C. diphteriae 3 - - Enterococcus spp. 9 - - E. coli 9 - - F. tularensis one - - H. influenzae 2 - - H. parainfluenzae one K. oxytoca 2 - - K. pneumoniae four L. monocitogenes 3 - - M. catarrhalis one - - P. shigelloides 3 - - P. mirabilis one - - P. vulgaris 3 P. aeruginosae 5 - - P. cepacia one P. maltophilia one S. paratyphi 3 - - S. typhi one S. typhimurium one S. marcescens 3 - - S. sonnei 3 - - S. flexneri 3 S. pyogenes four - - S. pneumoniae eleven S. salivarius one S. albus one - - S. aureus 9 S. epidermidis 5 S. pyogenes one Vibrio cholerae eltor 3 - - Y. enterocolitica 3 - - Y. pestis one Y. pseudotuberculosis one Candida spp. 5 - -

Table 4 Type of material Number of samples studied PCR-EF PCR-RHF Bacteriological IFA * IHA ** Sectional 2 + + + Unable to determine Unable to determine material: lung fragments tracheal fragments 2 + + + - "- - "- fragments 2 + + + - "- - "- head and spinal cord Sputum fifteen - - - - "- - "- BALL 3 - - - - "- - "- Smears from 9 - - - - "- - "- oropharynx Urine 5 - - Unable to determine - "- Blood serum 3 - - Unable to determine - Unable to determine Blood and serum samples, artificially infected with L. pneumophila Philadelphia 1 333151 ATCC four + + + + - "- Total: 45 45 45 37 7 5 Note: * - “A kit for the total detection of IgM-IgG antibodies to L. pneumophila serogroup 1-6” by Vircell (Spain). ** - Kit for detection of soluble antigen of L. pneumophila serogroup I in urine "Binax NOW Legionella Urinary Antigentest" (BINAX, USA). (-) - negative result. (+) - a positive result.

Table 5 Types of material Number of samples studied PCR-EF PCR-RHF Bacteriological Drinking water samples fourteen - - - Flushing from drinking water systems fifteen - - - Flushing from heating systems 10 - - - Flushing from air conditioning systems 12 - - - Samples of drinking water artificially infected with L. pneumophila Philadelphia 1 333151 ATCC 3 + + + Washings from the conditioner artificially infected with L. pneumophila Philadelphia 1 333151 ATCC 3 + + + Total: 57 57 57 57 Note: (-) - negative result; (+) - a positive result.

Table 6 Serogroup L. pneumophila Number of samples PCR-REF detection PCR-RHF detection one 3 + + 2 3 + + 3 3 + + four 3 + + 5 3 + + 6 3 + + 13 3 + + 2-14 * 10 + + Reference strains, CDC, (Atlanta, USA), 1988 L. dumoffii ATCC 33343 TEX-KL 3 L. micdadei 3 ATCC 33218 Tatlock L. pneumophila 3 + + Philadelphia 1 ATCC 33152 L. pneumophila 3 + + Togus 2 ATCC 33154 L. pneumophila 3 + + Bloomington 3 ATCC 33155 Note: * - the serogroup was determined using a set of reagents for latex agglutination NEW Legionella Latex Test Serogroup 1-14 (Oxoid, UK).

Claims (1)

A kit for the detection of the pathogen L. pneumophila in biological material and environmental objects containing species-specific primers Lp1 - 5'-GCTTGGTGCTGTGAAAGGTA-3 ', Lp2 - 5'-CCTCCAATAAAGGAGATGCAA-3' and probe Lp-s - 5'ACCGTG-TCG-TCGT-TCG-TCGT-TCG-TCGT-TCG-TCGTAC BHQ2-3 'used in amplification of a fragment of the L. pneumophila ftsZ gene within the range of 397 to 758 nucleotides of the complete sequence of a locus having the nucleotide sequence of SEQ ID NO 1.

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